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Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5 times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20 times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum. [ABSTRACT FROM AUTHOR]

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

The 20S proteasome core particle (20S CP) plays an integral role in cellular homeostasis by degrading proteins no longer required for function. The process is, in part, controlled via gating residues localized to the ends of the heptameric barrel-like CP structure that occlude substrate entry pores, preventing unregulated degradation of substrates that might otherwise enter the proteasome. Previously, we showed that the N-terminal residues of the α-subunits of the CP from the archaeon Thermoplasma acidophilum are arranged such that, on average, two of the seven termini are localized inside the lumen of the proteasome, thereby plugging the entry pore and functioning as a gate. However, the mechanism of gating remains unclear. Using solution NMR and a labeling procedure in which a series of mixed proteasome rings are prepared such that the percentage of gate-containing subunits is varied, we address the energetics of gating and establish whether gating is a cooperative process involving the concerted action of residues from more than a single protomer. Our results establish that the intrinsic probability of a gate entering the lumen favors the in state by close to 20-fold, that entry of each gate is noncooperative, with the number of gates that can be accommodated inside the lumen a function of the substrate entry pore size and the bulkiness of the gating residues. Insight into the origin of the high affinity for the in state is obtained from spin-relaxation experiments. More generally, our approach provides an avenue for dissecting interactions of individual protomers in homo-oligomeric complexes. [ABSTRACT FROM AUTHOR]

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

The main phospholipid (MPL) of Thermoplasma acidophilum DSM 1728 was isolated, purified and physico-chemically characterized by differential scanning calorimetry (DSC)/differential thermal analysis (DTA) for its thermotropic behavior, alone and in mixtures with other lipids, cholesterol, hydrophobic peptides and pore-forming ionophores. Model membranes from MPL were investigated; black lipid membrane, Langmuir-Blodgett monolayer, and liposomes. Laboratory results were compared to computer simulation. MPL forms stable and resistant liposomes with highly proton-impermeable membrane and mixes at certain degree with common bilayer-forming lipids. Monomeric bacteriorhodopsin and ATP synthase from Micrococcus luteus were co-reconstituted and light-driven ATP synthesis measured. This review reports about almost four decades of research on Thermoplasma membrane and its MPL as well as transfer of this research to Thermoplasma species recently isolated from Indonesian volcanoes.

Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and physiological studies. Mannonate dehydratases from archaea have not been studied in detail. Here, we identified and characterised the first archaeal mannonate dehydratase (TaManD) from the thermoacidophilic archaeon Thermoplasma acidophilum. The recombinant TaManD enzyme was optimally active at 65 °C and showed high specificity towards D-mannonate and its lactone, D-mannono-1,4-lactone. The gene encoding for TaManD is located adjacent to a previously studied mannose-specific aldohexose dehydrogenase (AldT) in the genome of T. acidophilum. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that the mannose-specific AldT produces the substrates for TaManD, demonstrating the possibility for an oxidative metabolism of mannose in T. acidophilum. Among previously studied mannonate dehydratases, TaManD showed closest homology to enzymes belonging to the xylose isomerase-like superfamily. Genetic analysis revealed that closely related mannonate dehydratases among archaea are not located in a hexuronate gene cluster like in bacteria, but next to putative aldohexose dehydrogenases, implying a different physiological role of mannonate dehydratases in those archaeal species.

Archaea contain membrane lipids that differ from those found in the other domains of life (Eukarya and Bacteria). These lipids consist of isoprenoid chains attached via ether bonds to the glycerol carbons at the sn -2,3 positions. Two types of ether lipids are known, polar diether lipids and bipolar tetraether lipids. The inherent chemical stability and unique membrane-spanning characteristics of tetraether lipids render them interesting for oral drug delivery purposes. Archaeal lipids form liposomes spontaneously (archaeosomes) and may be incorporated in conventional liposomes (mixed vesicles). Both types of liposomes are promising to protect their drug cargo, such as therapeutic peptides, against the acidic environment of the stomach and proteolytic degradation in the intestine. They appear to withstand lipolytic enzymes and bile salts and may thus deliver orally administered therapeutic peptides to distant sections of the intestine or to the colon, where they may be absorbed, eventually by the help of absorption enhancers. Archaeal lipids and their semisynthetic derivatives may thus serve as biological source for the next generation oral drug delivery systems. The aim of this review is to present a systematic overview over existing literature on archaea carrying diether and tetraether lipids, lipid diversity, means of lipid extraction and purification, preparation and in vitro stability studies of archaeal lipid-based liposomal drug carriers and in vivo proof-of concepts studies. [ABSTRACT FROM AUTHOR]

In organisms with circular chromosomes, such as bacteria and archaea, an odd number of homologous recombination events can generate a chromosome dimer. Such chromosome dimers cannot be segregated unless they are converted to monomers before cell division. In Escherichia coli, dimer-to-monomer conversion is mediated by the paralogous XerC and XerD recombinases at a specific dif site in the replication termination region. Dimer resolution requires the highly conserved cell division protein/chromosome translocase FtsK, and this site-specific chromosome resolution system is present or predicted in most bacteria. However, most archaea have only XerA, a homologue of the bacterial XerC/D proteins, but no homologues of FtsK. In addition, the molecular mechanism of XerA-mediated chromosome resolution in archaea has been less thoroughly elucidated than those of the corresponding bacterial systems. In this study, we identified two XerA-binding sites ( dif1 and dif2) in the Thermoplasma acidophilum chromosome. In vitro site-specific recombination assays showed that dif2, but not dif1, serves as a target site for XerA-mediated chromosome resolution. Mutational analysis indicated that not only the core consensus sequence of dif2, but also its flanking regions play important roles in the recognition and recombination reactions mediated by XerA. [ABSTRACT FROM AUTHOR]

The crystal structure of the Ta1207 protein from Thermoplasma acidophilum is reported. Ta1207 was identified in a screen for high-molecular-weight protein complexes in T. acidophilum. In solution, Ta1207 forms homopentamers of 188 kDa. The crystal structure of recombinant Ta1207 solved by Se-MAD at 2.4 Å resolution revealed a complex with fivefold symmetry. In the crystal lattice, calcium ions induce the formation of a nanocage from two pentamers. The Ta1207 protomers comprise two domains with the same novel α/β topology. A deep pocket with a binding site for a negatively charged group suggests that Ta1207 functions as an intracellular receptor for an unknown ligand. Homologues of Ta1207 occur only in Thermoplasmatales and its function might be related to the extreme lifestyle of these archaea. The thermostable Ta1207 complex might provide a useful fivefold-symmetric scaffold for future nanotechnological applications. [ABSTRACT FROM AUTHOR]

RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e. in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in theCMGcomplex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11- 3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN (GINS-associated nuclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2·TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM (T. acidophilum MCM) helicase in vitro, and the TaRecJ2·TaMCM·TaGINS complex was also observed in vivo. However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro. These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya. [ABSTRACT FROM AUTHOR]

Helicases are proposed to unwind dsDNA primarily by translocating on one strand to sterically exclude and separate the two strands. Hexameric helicases in particular have been shown to encircle one strand while physically excluding the other strand. In this article, we will detail experimental methods used to validate specific interactions with the excluded strand on the exterior surface of hexameric helicases. Both qualitative and quantitative methods are described to identify an excluded strand interaction, determine the exterior interacting residues, and measure the dynamics of binding. The implications of exterior interactions with the nontranslocating strand are discussed and include forward unwinding stabilization, regulation of the unwinding rate, and DNA damage sensing. [ABSTRACT FROM AUTHOR]

Cytosolic, globular droplets with an average diameter of 50 nm were observed in vitriied Thermoplasma acidophilum cells by means of cryo-electron tomography. These droplets were isolated by column chromatography and immunoprecipitation protein puriication methods. Subsequent chemical and biochemical analyses identiied lipid and protein components, respectively. Two major lipid components, comigrating menaquinones at the solvent front and the slower migrating Thermoplasma polar lipid U4, were detected by TLC experiments. The major protein component was identiied as the 153 amino acid long Ta0547 vitellogenin-N domain protein. This domain has been found so far exclusively in large lipid transport proteins of vertebrates and non-vertebrates. Blast protein database homology searches with Ta0547 did not return any eukaryal hits; homologous sequences were found only in thermo-acidophilic archaeons. However, a proile-sequence domain search performed with the vitellogenin-N domain (PF01347) hmm-proile against the T. acidophilum proteome returned Ta0547 as hit. Electron microscopy appearance of isolated droplets resembled to lipoprotein particles. However, no (tetraether) lipid layer could be detected on the droplets surface, rather hydrophobic compounds of the electron dense lumen were surrounded by a denser discontinuous protein boundary. Based on described features, these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet. [ABSTRACT FROM AUTHOR]

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97.We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded. [ABSTRACT FROM AUTHOR]

Site-specific Xer recombination plays a pivotal role in reshuffling genetic information. Here, we report the 2.5 Å crystal structure of XerA from the archaean Thermoplasma acidophilum. Crystallographic data reveal a uniquely open conformational state, resulting in a C-shaped clamp with an angle of ~ 48° and a distance of 57 Å between the core-binding and the catalytic domains. The catalytic nucleophile, Tyr264, is positioned in cis-cleavage mode by XerA's C-term tail that interacts with the CAT domain of a neighboring monomer without DNA substrate. Structural comparisons of tyrosine recombinases elucidate the dynamics of Xer recombinase. [ABSTRACT FROM AUTHOR]

We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach. [ABSTRACT FROM AUTHOR]

Thermoplasma acidophilum is a thermophilic archaeon that uses both non-phosphorylative Entner-Doudoroff (ED) pathway and Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. While triosephosphate isomerase (TPI), a well-known glycolytic enzyme, is not involved in the ED pathway in T. acidophilum, it has been considered to play an important role in the EMP pathway. Here, we report crystal structures of apo- and glycerol-3-phosphate-bound TPI from T. acidophilum (TaTPI). TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight β-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. We confirmed the dimeric conformation of TaTPI by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 as well as helix 4 of thermostable tetrameric TPIs have been known to play crucial roles in oligomerization, forming a hydrophobic interface. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent Td value of 74.6°C and maintains its overall structure with some changes in the secondary structure contents at extremely acidic conditions (pH 1–2). Based on our structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of Thermoplasma acidophilum TPI. [ABSTRACT FROM AUTHOR]

The glyceraldehyde dehydrogenase from Thermoplasma acidophilum ( TaAlDH) is a microbial enzyme that catalyzes the oxidation of D-glyceraldehyde to D-glycerate in the artificial enzyme cascade designed for the conversion of glucose to the organic solvents isobutanol and ethanol. Various mutants of TaAlDH were constructed by a random approach followed by site-directed and saturation mutagenesis in order to improve the properties of the enzyme that are essential for its functioning within the cascade. Two enzyme variants, wild-type TaAlDH ( TaAlDHwt) and an F34M+S405N variant ( TaAlDH F34M+S405N), were successfully crystallized. Crystals of TaAlDHwt belonged to the monoclinic space group P1211 with eight molecules per asymmetric unit and diffracted to a resolution of 1.95 Å. TaAlDH F34M+S405N crystallized in two different space groups: triclinic P1 with 16 molecules per asymmetric unit and monoclinic C121 with four molecules per asymmetric unit. These crystals diffracted to resolutions of 2.14 and 2.10 Å for the P1 and C121 crystals, respectively. [ABSTRACT FROM AUTHOR]

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. [ABSTRACT FROM AUTHOR]

Slaughterhouse waste is an energy rich feedstock suitable for anaerobic digestion processes. However, chemical characterization showed a deficiency in essential trace elements which are critical for optimal performance of the process. Hence this study investigated the degree of bioavailability of trace elements in four semi-continuous lab-scale AD tests accepting slaughterhouse waste under mesophilic conditions (38 °C) and a moderate organic loading rate of 2.2 kg/m 3 d. Parameters, such as volatile fatty acid (VFA) concentration, COD removal rate and specific methane yield were compared to the results of sequential extraction analysis. The highest methane yield (250–275 Nm 3 /t COD), lowest accumulation of VFA (<500 mg/l) and high COD removal rate (75–80%) was obtained when the total concentration of 11.4 mg/l Ni, 25.4 mg/l Co and 4.8 mg/l Mo was present in the reactor, of which 62% of Ni and Co, and 68% of Mo were bioavailable for microbial uptake. Based on these results it can be recommended that a supply of 2.5 g/t Ni, 3.5 g/t Co, 0.6 g/t Mo and 0.05 g/t Se provide optimal conditions for anaerobic digestion of slaughterhouse waste. [ABSTRACT FROM AUTHOR]

The article discusses a study related to the importance of disordered interdomain region of Gins for functional tetramer formation in Thermoplasma acidophilum to stimulate Multichip modules MCM helicase. Topics discussed include activation of the eukaryotic MCM by forming the CMG complex with Cdc45 and Gins, stimulation in the DNA-binding, ATPase, and helicase activities of TaMCM by interaction of TaGins51 protein with the MCM and necessity of MCM activation in formation of the Gins51 tetramer.

The article presents a high-resolution reconstruction of the Thermoplasma acidophilum 20S proteasome by using the cryo-electron microscopy (cryoEM) technique. According to the article, high-resolution enables in the identification of identifying the rotameric conformation by amino-acid side chains, or rotamers and are of interest in the drug industry.

We identified the non-phosphorylated l-rhamnose metabolic pathway (Rha_NMP) genes that are homologous to those in the thermoacidophilic archaeon Thermoplasma acidophilum in the genome of the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans. However, unlike previously known 2-keto-3-deoxy- l-rhamnonate ( l-KDR) dehydrogenase (KDRDH) which belongs to the short chain dehydrogenase/reductase superfamily, the putative KDRDHs in S. thermosulfidooxidans (Sulth_3557) and T. acidophilum (Ta0749) belong to the medium chain dehydrogenase/reductase (MDR) superfamily. We demonstrated that Sulth_3559 and Sulth_3557 proteins from S. thermosulfidooxidans function as l-rhamnose dehydrogenase and KDRDH, respectively. Sulth_3557 protein is an NAD-specific KDRDH with optimal temperature and pH of 50 °C and 9.5, respectively. The K and V values for l-KDR were 2.0 mM and 12.8 U/mg, respectively. Sulth_3557 also showed weak 2,3-butanediol dehydrogenase activity. Phylogenetic analysis suggests that Sulth_3557 and its homologs form a new subfamily in the MDR superfamily. The results shown in this study imply that thermoacidophilic archaea metabolize l-rhamnose to pyruvate and l-lactate by using the MDR-family KDRDH similarly to that of the thermoacidophilic bacterium S. thermosulfidooxidans. [ABSTRACT FROM AUTHOR]

We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m¹I, and m22G, which were not reported previously in this tRNA, and the m²G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m²G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed. [ABSTRACT FROM AUTHOR]

Asparaginases (ASNases) participate in the metabolism of all living organisms in the hydrolysis of free asparagines. Bacterial ASNases are used in cancer chemotherapy as they efficiently deplete amino acids. However, allergic reactions and silent inactivation represent critical limitations to their extended use. The rationale of this study was to identify, express, and characterize a plant-type L-ASNase from the archaeon Thermoplasma acidophilum as an enzyme with potentially improved characteristics. The Ta0338 orf was cloned into the pET28a(+) expression vector and overexpressed as a soluble protein with a molecular weight of 32 kDa. The quantity of recombinant L-ASNase produced in Escherichia coli was estimated as 9.68 mg/l. The purified protein showed evident autocatalytic processing of the zymogen at 4° and 37°C at physiological pH of 7.2 and clearly generated the expected alpha and beta subunits of 18 and 13 kDa, respectively. We propose that Ta-ASNase represents a potential biotechnological product for therapeutic purposes. [ABSTRACT FROM AUTHOR]

A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome. [ABSTRACT FROM AUTHOR]

The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active minicomplex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures. [ABSTRACT FROM AUTHOR]

Free methionine- R -sulfoxide reductase (fRMsr) enzymes only reduce the free form of methionine- R -sulfoxide and can be grouped into two types with respect to the number of conserved Cys residues in the active sites. In this work, the crystal structures of type II fRMsr from Thermoplasma acidophilum ( Ta fRMsr), which contains two conserved Cys, have been determined in native form and in a complex with the substrate. The overall structure of Ta fRMsr consists of a central β-sheet encompassed by a two-α-helix bundle flanking on one side and one small α-helix on the other side. Based on biochemical and growth complementation assays, Cys 84 is demonstrated to be the catalytic Cys. The data also show that Ta fRMsr functions as an antioxidant protein. Structural analyses reveal insights into substrate recognition and orientation, conformational changes in the active site during substrate binding, and the role of active site residues in substrate binding. A model for the catalytic mechanism of type II Ta fRMsr is suggested, in which intramolecular disulfide bond formation is not involved. In addition, the biochemical, enzymatic, and structural properties of type II Ta fRMsr are compared with those of type I enzymes. [ABSTRACT FROM AUTHOR]

In DNA replication studies, the mechanism for regulation of the various steps from initiation to elongation is a crucial subject to understand cell cycle control. The eukaryotic minichromosome maintenance (MCM) protein complex is recruited to the replication origin by Cdc6 and Cdt1 to form the pre-replication complex, and participates in forming the CMG complex formation with Cdc45 and GINS to work as the active helicase. Intriguingly, Thermoplasma acidophilum, as well as many other archaea, has only one Gins protein homolog, contrary to the heterotetramer of the eukaryotic GINS made of four different proteins. The Gins51 protein reportedly forms a homotetramer (TaGINS) and physically interacts with TaMCM. In addition, TaCdc6-2, one of the two Cdc6/Orc1 homologs in T. acidophilum reportedly stimulates the ATPase and helicase activities of TaMCM in vitro. Here, we found a reaction condition, in which TaGINS stimulated the ATPase and helicase activities of TaMCM in a concentration dependent manner. Furthermore, the stimulation of the TaMCM helicase activity by TaGINS was enhanced by the addition of TaCdc6-2. A gel retardation assay revealed that TaMCM, TaGINS, and TaCdc6-2 form a complex on ssDNA. However, glutaraldehyde-crosslinking was necessary to detect the shifted band, indicating that the ternary complex of TaMCM-TaGINS-TaCdc6-2 is not stable in vitro. Immunoprecipitation experiment supported a weak interaction of these three proteins in vivo. Activation of the replicative helicase by a mechanism including a Cdc6-like protein suggests the divergent evolution after the division into Archaea and Eukarya. [ABSTRACT FROM AUTHOR]

The lack of a few conserved enzymes in the classical mevalonate pathway and the widespread existence of isopentenyl phosphate kinase suggest the presence of a partly modified mevalonate pathway in most archaea and in some bacteria. In the pathway, (R)-mevalonate 5-phosphate is thought to be metabolized to isopentenyl diphosphate via isopentenyl phosphate. The long anticipated enzyme that catalyzes the reaction from (R)-mevalonate 5-phosphate to isopentenyl phosphate was recently identified in a Cloroflexi bacterium, Roseiflexus castenholzii, and in a halophilic archaeon, Haloferax volcanii. However, our trial to convert the intermediates of the classical and modified mevalonate pathways into isopentenyl diphosphate using cell-free extract from a thermophilic archaeon Thermoplasma acidophilum implied that the branch point intermediate of these known pathways, i.e. (R)-mevalonate 5-phosphate, is unlikely to be the precursor of isoprenoid. Through the process of characterizing the recombinant homologs of mevalonate pathway-related enzymes from the archaeon, a distant homolog of diphosphomevalonate decarboxylase was found to catalyze the phosphorylation of (R)-mevalonate to yield (R)-mevalonate 3-phosphate. The product could be converted into isopentenyl phosphate, probably through (R)-mevalonate 3,5-bisphosphate, by the action of unidentified T. acidophilum enzymes fractionated by anion-exchange chromatography. These findings demonstrate the presence of a third alternative Thermoplasmatype mevalonate pathway, which involves (R)-mevalonate 3-phosphotransferase and probably both (R)-mevalonate 3-phosphate 5-phosphotransferase and (R)-mevalonate 3,5-bisphosphate decarboxylase, in addition to isopentenyl phosphate kinase. [ABSTRACT FROM AUTHOR]

Abstract: Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60 °C and pH 6, and the enzyme exhibited notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives ethanol, EDTA, urea, DTT, SDS, β-mercaptoethanol, methanol, isopropanol and n-butanol. Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases. [Copyright &y& Elsevier]

The iron-dependent regulator (IdeR) is a metal ion-activated transcriptional repressor that regulates the expression of genes encoding proteins involved in iron uptake to maintain metal-ion homeostasis. IdeR is a functional homologue of the diphtheria toxin repressor (DtxR), and both belong to the DtxR/MntR family of metalloregulators. The structure of Fe2+-bound IdeR (TA0872) from Themoplasma acidophilum was determined at 2.1 Å resolution by X-ray crystallography using single-wavelength anomalous diffraction. The presence of Fe2+, which is the true biological activator of IdeR, in the metal-binding site was ascertained by the use of anomalous difference electron-density maps using diffraction data collected at the Fe absorption edge. Each DtxR/IdeR subunit contains two metal ion-binding sites separated by 9 Å, labelled the primary and ancillary sites, whereas the crystal structures of IdeR from T. acidophilum show a binuclear iron cluster separated by 3.2 Å, which is novel to T. acidophilum IdeR. The metal-binding site analogous to the primary site in DtxR was unoccupied, and the ancillary site was occupied by binuclear clustered ions. This difference suggests that T. acidophilum IdeR and its closely related homologues are regulated by a mechanism distinct from that of either DtxR or MntR. T. acidophilum IdeR was also shown to have a metal-dependent DNA-binding property by electrophoretic mobility shift assay. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the current work is to develop an environmentally friendly process for the removal of heavy metals (Cu, Zn, Ni, Cd, Al, Cr, Pb) from recycling industry electronic waste with a consortium of Sulfobacillus thermosulfidooxidans and Thermoplasma acidophilum. The performances of commercial S0 powder and biogenic S0 sludge as substrates for the bio-removal of heavy metals were compared. Empirical models for the bioleaching process based on a statistical analysis were developed to evaluate the individual and combined effects of critical variables including S0 dosage, particle size, pulp density and bacterial feed formulation (inoculum size and inoculation style) in shaken flasks while specifying the effective variable ranges. Upscale feasibility experiments in a stirred tank reactor demonstrated a maximum metal bio-removal efficiency (92%) at a 1% dosage of biogenic S0 sludge and 2% dosage of commercial S0 powder (82%), given a 15% pulp density and 150 μm particle size with an intermittent low-concentration addition of inoculum (1ｘ106 cells/mL). Biogenic S0 sludge showed a higher degree of S0 oxidation (95%) in a shorter time period (12 days) compared to commercial S0 powder (82% in 24 days), thereby reducing the process cost. Risk assessments of discarded electronic wastes before and after bioremediation by the toxicity characteristic leaching procedure (TCLP), waste extraction test (WET), synthetic precipitation test (SPLP) and total threshold limit concentration (TTLC) indicated that the leaching/toxicity of bio-remediated residue was well within the regulatory limits. [Copyright &y& Elsevier]

The 20S core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. Regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. Although atomic resolution models of both open and closed states of the proteasome have been elucidated, the mechanism by which gates exchange between these states remains to be understood. Here, this is investigated by using magnetization transfer NMR spectroscopy focusing on the 20S proteasome core particle from Thermoplasma acidophilum. We show from viscosity-dependent proteasome gating kinetics that frictional forces originating from random solvent motions are critical for driving the gating process. Notably, a small effective hydrodynamic radius (EHR; <4Å) is obtained, providing a picture in which gate exchange proceeds through many steps involving only very small segment sizes. A small EHR further suggests that the kinetics of gate interconversion will not be affected appreciably by large viscogens, such as macromolecules found in the cell, so long as they are inert. Indeed, measurements in cell lysate reveal that the gate interconversion rate decreases only slightly, demonstrating that controlled studies in vitro provide an excellent starting point for understanding regulation of 20S core particle function in complex, biologically relevant environments. [ABSTRACT FROM AUTHOR]

Mini-chromosome maintenance (MCM) proteins are the replicative helicase necessary for DNA replication in both eukarya and archaea. Most of archaea only have one MCM gene. Here, we report a 1.8-Å crystal structure of the N-terminal MCM from the archaeon Thermoplasma acidophilum (tapMCM). In the structure, the MCM N-terminus forms a right-handed filament that contains six subunits in each turn, with a diameter of 25Å of the central channel opening. The inner surface is highly positively charged, indicating DNA binding. This filament structure with six subunits per turn may also suggests a potential role for an open-ring structure for hexameric MCM and dynamic conformational changes in initiation and elongation stages of DNA replication. [ABSTRACT FROM AUTHOR]

A new method is presented for synthesizing arbutin glycosides using α-glucosidase (AglA) from Thermoplasma acidophilum and its glycosynthase mutant. An α-glycosynthase was constructed by substituting the catalytic nucleophile with the non-nucleophile glycine. Enzyme activity was then recovered using an external nucleophile. The transglycosylation reaction of AglA using maltose as a donor and arbutin as an acceptor produced arbutin coupled with a glucose moiety. The products were isolated and further analysed using preparative recycling HPLC. Arbutin glycosides linked to C-3, C-4, and C-6 were identified using NMR. The transglycosylation products of AglA were used as substrates for the enzyme reaction, which were hydrolyzed back again and reduced final yields. The glycosynthase mutant produced one main arbutin glycoside linked to C-4 with a yield of 38 % without further observed hydrolysis. [ABSTRACT FROM AUTHOR]

Abstract: We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available. [Copyright &y& Elsevier]

The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as nanoreactor for atom-transfer radical polymerization (ATRP). A copper catalyst was entrapped into the THS to confine the polymerization into this protein cage. THS possesses pores that are wide enough to release polymers into solution. The nanoreactor favorably influenced the polymerization of N-isopropyl acrylamide and poly(ethylene glycol)methylether acrylate. Narrowly dispersed polymers with polydispersity indices (PDIs) down to 1.06 were obtained in the protein nanoreactor, while control reactions with a globular protein-catalyst conjugate only yielded polymers with PDIs above 1.84. [ABSTRACT FROM AUTHOR]