Sulfolobus solfataricus

A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli . The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N -acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electr...

Using Sso7d from Sulfolobus solfataricus as the DNA binding protein fused to Taq DNA polymerase at its amino terminus, we report the hyper-expression and a novel purification methodology of Sso7d-Taq polymerase (S-Taq) using aqueous two-phase extraction system followed by Ni-affinity chromatography. The utility of such a fusion enzyme in carrying out PCR of human genes from whole blood directly and in detecting hepatitis B virus from clinical samples is demonstrated in this article. We present data on the enhanced thermo-stability of S-Taq DNA polymerase over Taq DNA polymerase and also provide evidence of its higher stability with detergents in comparison to Taq polymerase. The purified S-Taq protein showed acceptable limits of host genomic DNA levels without the use of DNases and other DNA precipitating agents and shows promising potential for use in PCR based diagnostics, in-situ PCR's and forensic science.

All living organisms need a DNA replication mechanism and it has been conserved in the three domains of life throughout evolutionary process. Primase is the enzyme responsible for synthesizing de novo RNA primers in DNA replication. Archaeo-Eukaryotic Primase (AEP) is the superfamily that typically forms a heterodimeric complex containing both a small catalytic subunit (PriS) and a large accessory noncatalytic subunit (PriL). Sulfolobus solfataricus is a model organism for research on the Genetics field. The aim of this work was to evaluate, via Bioinformatics tools, three mutations in the large subunit (PriL) of the archaeon Sulfolobus solfataricus. The aspartic acid residue in the positions (Asp) 62, (Asp) 235, (Asp) 241 have been substituted by glutamic acid (Glu). The highest positive free energy variation of the three substitutions analyzed occurred with the mutation at the (Asp) 241 site. The in silico analysis suggested that these mutations in PriL may destabilize its tridimensional structure interfering with replication mechanisms of Sulfolobus solfataricus. Moreover, it may also alter interactions with other molecules, making salt bridges, for instance.

Over 40 human diseases are associated with the formation of well-defined proteinaceous fibrillar aggregates. Since the oligomers precursors to the fibrils are increasingly recognized to be the causative agents of such diseases, it is important to elucidate the mechanism of formation of these early species. The acylphosphatase from Sulfolobus solfataricus is an ideal system as it was found to form, under conditions in which it is initially native, two types of prefibrillar aggregates: (1) initial enzymatically active aggregates and (2) oligomers with characteristics reminiscent of amyloid protofibrils, with the latter originating from the structural reorganization of the initial assemblies. By studying a number of protein variants with a variety of biophysical techniques, we have identified the regions of the sequence and the driving forces that promote the first aggregation phase and show that the second phase consists in a cooperative conversion involving the entire globular fold.

Protein p3, a ribonuclease we previously isolated from the archaebacterium Sulfolobus solfataricus [P. Fusi et al. (1993) Eur. J. Biochem. 211,305-310], was subjected to complete amino acid sequencing. It consisted of 75 residues, with a calculated Mr of 8582, a pI of 10.1, and had some degree of monomethylation at Lys-4 and Lys-6. p2, a previously sequenced, 62-residue ribonuclease from the same organism, had an identical sequence for 57 consecutive residues starting from the N-terminus. p2 and p3 also showed a striking similarity to five other proteins previously isolated from Sulfolobus strains and identified as DNA-binding proteins. However, the C-terminus, 10 residue region of p3 did not show any similarity to these proteins; in contrast, it was significantly similar to stretches in three eubacterial ribonucleases from Bacillus strains. No difference between p2 and p3 has so far been detected as regards their catalytic properties. Available data suggest that these molecules have a narrow substrate specificity and probably play specific roles in RNA processing.

Sso7d from the thermoacidophilic archaebacterium Sulfolobus solfataricus is a small globular protein with a known three-dimensional structure. Inspection of the structure reveals that Phe31 is a member of the aromatic cluster forming the protein hydrophobic core, whereas Trp23 is located on the protein surface and its side chain exposed to the solvent. The thermodynamic consequences of the substitution of these two residues in Sso7d have been investigated by comparing the temperature-induced denaturation of Sso7d with that of three mutants: F31A-Sso7d, F31Y-Sso7d, and W23A-Sso7d. The denaturation processes proved to be reversible for all proteins, and represented well by the two-state N S D transition model in a wide range of pH. All three mutants are less thermally stable than the parent protein; in particular, in the pH range of 5.0-7.0, the F31A substitution leads to a decrease of 24 °C in the denaturation temperature, the F31Y substitution to a decrease of 10 °C, and the W23A substitution to a decrease of 6 °C. A careful thermodynamic analysis of such experimental data is carried out.

Background: Superparamagnetic iron oxide nanoparticles (MNP) offer several advantages for applications in biomedical and biotechnological research. In particular, MNP-based immobilization of enzymes allows high surface-to-volume ratio, good dispersibility, easy separation of enzymes from the reaction mixture, and reuse by applying an external magnetic field. In a biotechnological perspective, extremophilic enzymes hold great promise as they often can be used under non-conventional harsh conditions, which may result in substrate transformations that are not achievable with normal enzymes. This prompted us to investigate the effect of MNP bioconjugation on the catalytic properties of a thermostable carboxypeptidase from the hyperthermophilic archaeon Sulfolobus solfataricus (CPSso), which exhibits catalytic properties that are useful in synthetic processes. Results: CPSso was immobilized onto silica-coated iron oxide nanoparticles via NiNTA-His tag site-directed conjugation. Following the immobilization, CPSso acquired distinctly higher long-term stability at room temperature compared to the free native enzyme, which, in contrast, underwent extensive inactivation after 72 h incubation, thus suggesting a potential utilization of this enzyme under low energy consumption. Moreover, CPSso conjugation also resulted in a significantly higher stability in organic solvents at 40°C, which made it possible to synthesize N-blocked amino acids in remarkably higher yields compared to those of free enzyme. The nanobioconjugate of CPSso immobilized on silica-coated magnetic nanoparticles exhibited enhanced stability in aqueous media at room temperature as well as in different organic solvents. The improved stability in ethanol paves the way to possible applications of immobilized CPSso, in particular as a biocatalyst for the synthesis of N-blocked amino acids. Another potential application might be amino acid racemate resolution, a critical and expensive step in chemical synthesis.

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a b-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 3608 clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP-PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors.

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The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3ʹ,5ʹ-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A.

Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple (32)P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent (32)P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other comp...

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Silenced cells exhibit a reduced or peeled-off S-layer lattice, cell shape alterations and decreased surface glycosylation. These cells barely propagate but increase in diameter and DNA content, indicating impaired cell division; their phenotypes can be rescued through genetic complementation. Furthermore, S-layer depleted cells are less susceptible to infection with the virus SSV1. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is...

5'-Deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants were catalyzed efficiently by MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of ΔHcal (850kcal/mol) and Tmax (39°C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200-Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the di...

The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%-49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.

1-Nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG AP ). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dG AP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dG AP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases. Environmental pollutants have been shown to impact human health at the molecular level. One detrimental route is the mod-ification of genomic DNA and nucleotides (1). If DNA lesions are not recognized and removed by the cellular DNA repair machinery, they will stall replicative DNA polymerases (2-8). To rescue DNA replication, cells employ lesion bypass DNA polymerases to traverse unrepaired lesions. Most of these enzymes belong to the Y-family of DNA polymerases. The Y-family enzymes possess relatively flexible and solvent-accessible active sites to accommodate bulky DNA lesions (9, 10). However, Y-family DNA polymerases catalyze DNA synthesis over undamaged DNA with low fidelity and poor processivity (6, 10 -12). The Y-family DNA polymerases have been identified in all three domains of life, e.g. four in humans (DNA polymerases , , , and Rev1), two in Escherichia coli (DNA polymerases IV and V), and one in Sulfolobus solfataricus (Dpo4). Because Dpo4 can be expressed in E. coli and purified with a high yield, it has been extensively studied in vitro as a prototype Y-family enzyme. Dpo4 catalyzes DNA synthesis on an undamaged DNA template with a fidelity of one error per 1,000 -10,000 nucleotide incorporations based on presteady-state kinetic analysis from 37 to 56 °C (13-15). Dpo4 is capable of bypassing a myriad of DNA lesions including apurinic/apyrimidinic (abasic) sites (16 -19), 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (20, 21), 1,N 2 -etheno(⑀)guanosine ( ), cis-syn thymine-thymine dimer (23-25), cisplatin-induced 1,2-intrastrand cross-links with adjacent deoxyguanosines (cisplatin-d(GpG) adducts) , benzo[a]pyrene diol epoxide (BPDE) 5 on deoxyguanosine (BPDE-dG) or deoxyadenosine (BPDE-dA) (27, 28), and N-2-acetyl-aminofluorene (AAF) on deoxyguanosine (AAF-dG) (24). So far there are no comprehensive in vitro studies of the bypass of 1-nitropyrene (1-NP)-induced DNA adducts catalyzed by a Y-family DNA polymerase. 1-NP, one of the most abundant polycyclic aromatic hydrocarbons, is a product of incomplete diesel and gasoline combustion . There are two known pathways by which 1-NP is metabolized: nitro reduction (Scheme 1) and C-hydroxylation. When an aromatic ring of 1-NP is oxidized into non-DNA-reactive metabolites by

Sulfolobus solfataricusDNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C.

The realization that majority of microbes are not amenable to cultivation as isolates under laboratory conditions has led to the culture-independent metagenomic approach as a novel technique for novel biocatalyst discovery. A leachate fosmid shotgun metagenome library was constructed and subsequently screened for esterolytic activities on a tributyrin agar medium. Nucleotide sequencing and translational analysis of an esterase-positive fosmid clone led to the identification of a 1,281 bp esterase gene (estC) encoding a protein (EstC) of 427 aa with translated molecular weight of 46.3 kDa. The EstC primary structure contained a signal leader peptide (29 aa), which could be cleaved to form a mature protein of 398 aa with molecular weight 43.3 kDa. Homology searches revealed that EstC belonged to the family VIII esterases, which exploit a serine residue within the S-x-x-K motif as a catalytic nucleophile. Substrate specificity studies showed that EstC prefers short to medium acyl chain length of p-nitrophenyl esters, a characteristic typical of "true" carboxylesterases. Moreover, EstC represents the first member of the family VIII esterases with a leader peptide and a detectable promiscuous β-lactam hydrolytic activity. Site-directed mutagenesis studies also revealed that in addition to Ser103 and Lys106 residues, the Tyr219 residue also plays a catalytic role in EstC. The organic solvent stability and the specificity towards esters of tertiary alcohols linalyl acetate (3,7-dimethyl-1,6-octadien-3-yl acetate) make EstC potentially useful in biocatalysis.

Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N 2 -position of guanine to produce N 2 -BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2′-deoxy-N-[(7R,8S,9R,10S)-7,8,9,10-tetrahydro-7,8,9trihydroxybenzo[a]pyren-10-yl]-guanosine and 2′-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deaza-dATP, 2′-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson-Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the *

The effects of temperature and SDS on the three-dimensional organization and secondary structure of β-glycosidase from the thermophilic archaeon Sulfolobus solfataricus were investigated by CD, IR spectroscopy and differential scanning calorimetry. CD spectra in the near UV region showed that the detergent caused a remarkable change in the protein tertiary structure, and Abbreviations used : amide Ih, amide I band in 2 H 2 O medium ; CAPS, 3-(cyclohexylamino)propane-1-sulphonic acid ; DSC, differential scanning calorimetry ; FT-IR, Fourier transform IR ; Sβgly, Sulfolobus solfataricus β-glycosidase.

The role of the extra helix do in the N-terminal extension of the eight-fold ßa barrel of indoleglycerol phosphate synthase was probed by point mutation and truncation. Replacing invariant leucine 5 by valine of the enzyme from Escherichia coli affected neither k cat nor K M , but deletion of 8 N-terminal residues decreased solubility strongly. The similarly truncated variant from the hyperthermophile Sulfolohus solfataricus was soluble, and had the same k cat value as the wild-type protein but a 220-fold greater K M value. These results suggest that the Nterminal portion of helix Oo provides for strong binding of the substrate, but is not essential for stabilizing the bound transition state. Thus, three enzymes of tryptophan biosynthesis operate essentially as canonical eight-fold ßa barrels, as required for their divergent evolution.

Generation of the 3′ overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3′ single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus. We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when ...

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg 2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins ar...

The hyperthermostable b-glycosidases from the Archaea Sulfolobus solfataricus (SsbGly) and Pyrococcus furiosus (CelB) hydrolyse b-glycosides of d-glucose or d-galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and SsbGly show a marked preference for making new b(133) and b(136) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 times that of SsbGly and yields b-d-Galp-(136)-d-Glc and b-d-Galp-(133)-d-Glc in a molar ratio of < 1 : 2. The partitioning of galactosylated SsbGly between reaction with sugars [k Nu (m 21 ´s21 )] and reaction with water [k water (s 21 )] is about twice that of CelB. It gives a mixture of linear b-d-glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are b(136) and b(133) for the reactions catalysed by SsbGly and CelB, respectively. The accumulation of b-d-Galp-(136)-d-Glc at the end of lactose hydrolysis reflects a 3±10-fold specificity of both enzymes for the hydrolysis of b(133) over b( ) linked glucosides. Galactosyl transfer from SsbGly or CelB to d-glucose occurs with partitioning ratios, k Nu /k water , which are seven and . 170 times those for the reactions of the galactosylated enzymes with 1-propanol and 2-propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new b-glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate-limiting first-order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by < 2.5 log 10 units from structure±reactivity correlations based on the pK a of the leaving group.

To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H(2)O(2)) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 microM H(2)O(2). Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had sig...

The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host systems of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled six times over a 72 h period...

One of the outstanding questions in biology today is the origin of viruses. We have discovered a protein in the hyperthermophile Sulfolobus solfataricus while following proteome regulation during viral infection that led to the discovery of a fossil provirus. Characterization of the wild type and recombinant protein revealed that it assembled into virus-like particles with a diameter of ~32 nm. Sequence and structural analyses showed that the likely proviral capsid protein, Sso2749, is homologous to a protein from Pyrococcus furiosus that forms virus-like particles using the HK-97 major capsid protein fold. The SsP2-provirus appears mosaic and contains proteins with similarity to, among others, eukaryotic herpesviruses and tailed dsDNA bacteriophage families, reinforcing the hypothesis of a common ancestral gene pool across all three domains of life. This is the first description of the HK-97 fold in a crenarchaeal virus and the first direct genomic connection of linocin-like protein cages to a virus.

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses, however, a major exception are viruses with archaeal hosts. Archaeal virushost systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homolog in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

Background: The current paradigm of intracellular redox chemistry maintains that cells establish a reducing environment maintained by a pool of small molecule and protein thiol to protect against oxidative damage. This strategy is conserved in mesophilic organisms from all domains of life, but has been confounded in thermophilic organisms where evidence suggests that intracellular proteins have abundant disulfides. Methods: Chemical labeling and 2-dimensional gel electrophoresis were used to capture disulfide bonding in the proteome of the model thermophile Sulfolobus solfataricus. The redox poise of the metabolome was characterized using both chemical labeling and untargeted liquid chromatography mass spectrometry. Gene annotation was undertaken using support vector machine based pattern recognition. Results: Proteomic analysis indicated the intracellular protein thiol of S. solfataricus was primarily in the disulfide form. Metabolic characterization revealed a lack of reduced small molecule thiol. Glutathione was found primarily in the oxidized state (GSSG), at relatively low concentration. Combined with genetic analysis, this evidence shows that pathways for synthesis of glutathione do exist in the archaeal domain. Conclusions: In observed thermophilic organisms, thiol abundance and redox poise suggest that this system is not directly utilized for protection against oxidative damage. Instead, a more oxidized intracellular environment promotes disulfide bonding, a critical adaptation for protein thermostability. General significance: Based on the placement of thermophilic archaea close to the last universal common ancestor in rRNA phylogenies, we hypothesize that thiol-based redox systems are derived from metabolic pathways originally tasked with promoting protein stability.

The coordinates of the Pf Csx1 structure together with the corresponding structure factors have been deposited in the Protein Data Bank with the accession code of 4EOG. Conflicts of interest: The authors declare that they have no conflicts of interest with respect to this study.

A tri‐functional monomeric primase–polymerase domain encoded by the plasmid pIT3 from Sulfolobus solfataricus strain IT3 was identified using a structural–functional approach. The N‐terminal domain of the pIT3 replication protein encompassing residues 31–245 (i.e. Rep245) was modeled onto the crystallographic structure of the bifunctional primase–polymerase domain of the archaeal plasmid pRN1 and refined by molecular dynamics in solution. The Rep245 protein was purified following overexpression in Escherichia coli and its nucleic acid synthesis activity was characterized. The biochemical properties of the polymerase activity such as pH, temperature optima and divalent cation metal dependence were described. Rep245 was capable of utilizing both ribonucleotides and deoxyribonucleotides for de novo primer synthesis and it synthesized DNA products up to several kb in length in a template‐dependent manner. Interestingly, the Rep245 primase–polymerase domain harbors also a terminal nucleo...

-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA-and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars&#39; conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87°C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process.

Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87°C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process.

Maltooligosyltrehalose trehalohydrolase (MTHase) is an industrial enzyme for the production of trehalose. A DNA fragment of 1680 bp encoding for MTHase was cloned from Sulfobolus solfataricus DSM 1616 then fused with promoter acoA-amyE already amplified from pMSE3 vector by PCR to generate an expression cassette acoMTH. Afterward the cassette was inserted into pAC7 vector for expression of the gene in Bacillus subtilis WB800 – a conventional expression system. Gene MTH was inserted into the genome of B. subtilis WB800 by cross-exchange event of pAC7 vector with the host genome for expression of high quality and high quantity of extracellular recombinant protein. By crossing-exchange event at 3’amyE-5’amyE, the expressional cassette was integrated into B. subtilis WB800 genome. The expressional cassette was integrated into B. subtilis WB800 genome replacing 3’amyE-5’amyE, hindering the native amylase activity of the host. Expression of expected protein was confirmed by electrophoresi...

SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a β-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π-sugar interaction and reveal a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to ...

In Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (α, β, and γ), is pivotal for binding of charged initiator tRNA to the small ribosomal subunit. The crystal structure of the full-sized heterotrimeric aIF2 from Sulfolobus solfataricus in the nucleotidefree form has been determined at 2.8-Å resolution. Superposition of four molecules in the asymmetric unit of the crystal and the comparison of the obtained structures with the known structures of the aIF2αγ and aIF2βγ heterodimers revealed high conformational flexibility in the αand βsubunits. In fact, the full-sized aIF2 consists of a rigid central part, formed by the γ-subunit, domain 3 of the α-subunit, and the N-terminal α-helix of the β-subunit, and two mobile "wings," formed by domains 1 and 2 of the α-subunit, the central part, and the zinc-binding domain of the β-subunit. High structural flexibility of the wings is probably required for interaction of aIF2 with the small ribosomal subunit. Comparative analysis of all known structures of the γ-subunit alone and within the heterodimers and heterotrimers in nucleotide-bound and nucleotide-free states shows that the conformations of switch 1 and switch 2 do not correlate with the assembly or nucleotide states of the protein.

The recombinant b-glycosidase (EcSbgly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70°C). Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available b-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor. The results of the biotrasformation at 70°C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction. Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate. The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h − 1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h − 1). For the hydrolysis of the oleuropein by bioreactor at 60°C we used lower molar ratio oleuropein/enzyme only by the single step approach. In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h). The stability of the bioreactor at the operative

Background Archaea are a vast and unexplored domain. Bioinformatic techniques might enlighten the path to a higher quality genome annotation in varied organisms. Promoter sequences of archaea have the action of a plethora of proteins upon it. The conservation found in a structural level of the binding site of proteins such as TBP, TFB, and TFE aids RNAP-DNA stabilization and makes the archaeal promoter prone to be explored by statistical and machine learning techniques. Results and discussions In this study, experimentally verified promoter sequences of the organisms Haloferax volcanii, Sulfolobus solfataricus, and Thermococcus kodakarensis were converted into DNA duplex stability attributes (i.e. numerical variables) and were classified through Artificial Neural Networks and an in-house statistical method of classification, being tested with three forms of controls. The recognition of these promoters enabled its use to validate unannotated promoter sequences in other organisms. As ...

Highlights-Continuous biocatalytic membrane reactor (BMR) to hydrolyse organophosphates-Significant improvement of phosphotriesterase stability respect to the free enzyme-High organophosphate degradation (90%) by the BMR-Long term stability of the BMR (1 year) ABSTRACT Organophosphates (OPs) are highly toxic compounds used as pesticides and nerve agents. The devastating effects, reported in different studies, on the environment and human health indicate a serious scenario for both instantaneous and long terms effects. Bio-based strategies for OPs degradation seem the most promising solutions, particularly when extremophiles enzymes are used. These systems permit OPs degradation with high efficiency and specificity under mild conditions. However, as frequently observed, enzymes can easily lose activity in batch systems, so that a strategy to improve biocatalyst stability is highly needed, in order to develop continuous systems. In this work, for the first time, a continuous biocatalytic system for organophosphates (OPs) detoxification has been proposed by using a triple mutant of the thermostable phosphotriesterase (named SsoPox) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. The enzyme was covalently immobilized on polymeric membranes to develop a biocatalytic membrane reactor (BMR) able to hydrolyse a pesticide (paraoxon) contained in water. High paraoxon degradation (about 90%) and long term stability (1 year) were obtained when the enzyme was covalently immobilized on hydrophilic membranes. On the contrary, the enzyme in batch system completely loses its activity within few months after its solubilisation in buffer.

Cytochrome P450 from thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st) has been expressed in Escherichia coli and purified at high homogeneity. P450st was crystallized in an orthorhombic system with the space group P 2 1 2 1 2 1 and cell dimensions of a ¼ 53:6 A, b ¼ 55:1 A, and c ¼ 130:9 A, and the structure was determined at a 3.0 A resolution. The final R-factor was 0.194 (R free ¼ 0:235). Structural comparison with cytochrome P450 from S. solfataricus (CYP119) suggests that the region composed of the F to G helices and the Cl À binding site is responsible for the affinity for a ligand coordinating heme iron. Direct electrochemistry of P450st in a didodecyldimethylammonium bromide (DDAB) film on a plastic formed carbon (PFC) electrode has also been demonstrated. A quasi-reversible redox response has been observed even at elevated temperatures of up to 80°C.

, the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoGC base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain-DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain-phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.

Highlights-Continuous biocatalytic membrane reactor (BMR) to hydrolyse organophosphates-Significant improvement of phosphotriesterase stability respect to the free enzyme-High organophosphate degradation (90%) by the BMR-Long term stability of the BMR (1 year) ABSTRACT Organophosphates (OPs) are highly toxic compounds used as pesticides and nerve agents. The devastating effects, reported in different studies, on the environment and human health indicate a serious scenario for both instantaneous and long terms effects. Bio-based strategies for OPs degradation seem the most promising solutions, particularly when extremophiles enzymes are used. These systems permit OPs degradation with high efficiency and specificity under mild conditions. However, as frequently observed, enzymes can easily lose activity in batch systems, so that a strategy to improve biocatalyst stability is highly needed, in order to develop continuous systems. In this work, for the first time, a continuous biocatalytic system for organophosphates (OPs) detoxification has been proposed by using a triple mutant of the thermostable phosphotriesterase (named SsoPox) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. The enzyme was covalently immobilized on polymeric membranes to develop a biocatalytic membrane reactor (BMR) able to hydrolyse a pesticide (paraoxon) contained in water. High paraoxon degradation (about 90%) and long term stability (1 year) were obtained when the enzyme was covalently immobilized on hydrophilic membranes. On the contrary, the enzyme in batch system completely loses its activity within few months after its solubilisation in buffer.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA–DNA and SegB–DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon–helix–helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA–SegB–DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure ...

Differentiation of binding accurate DNA replication polymerases over error prone DNA lesion bypass polymerases is essential for the proper maintenance of the genome. The hyperthermophilic archaeal organism, Sulfolobus solfataricus (Sso), contains both a B-family replication (Dpo1) and a Y-family repair (Dpo4) polymerase and serves as a model system for understanding molecular mechanisms and assemblies for DNA replication and repair protein complexes. Protein crosslinking, isothermal titration calorimetry, and analytical ultracentrifugation have confirmed a previously unrecognized dimeric Dpo4 complex bound to DNA. Binding discrimination between these polymerases on model DNA templates is complicated by the fact that multiple oligomeric species are influenced by concentration and temperature. Temperature dependent fluorescence anisotropy equilibrium binding experiments were used to separate discrete binding events for formation of trimeric Dpo1 and dimeric Dpo4 complexes on DNA. The associated equilibria are found to be temperature dependent, generally leading to improved binding at higher temperatures for both polymerases. At high temperatures, DNA binding by Dpo1 monomer is favored over Dpo4 monomer, but binding of Dpo1 trimer is even more strongly favored over Dpo4 dimer, thus providing thermodynamic selection. Greater processivities of nucleotide incorporation for trimeric Dpo1 and dimeric Dpo4 are also observed at higher temperatures, providing biochemical validation for the influence of tightly bound oligomeric polymerases. These results separate, quantify, and confirm individual and sequential processes leading to formation of oligomeric Dpo1 and Dpo4 assemblies on DNA and provide for a concentration and temperature dependent discrimination of binding undamaged DNA templates at physiological temperatures.

A thermostable thioredoxin reductase isolated from Sulfolobus solfataricus (SsTrxR) has been successfully crystallized in the absence and in the presence of NADP. Two different crystal forms have been obtained. Crystals of the form that yields higher resolution data (1.8 Å) belong to space group P2 1 2 1 2 1 , with unitcell parameters a = 76.77, b = 120.68, c = 126.85 Å. The structure of the enzyme has been solved by MAD methods using the anomalous signal from the Se atoms of selenomethionine-labelled SsTrxR. crystallization communications Acta Cryst. (2005). F61, 906-909 Ruggiero et al. Thioredoxin reductase 909

The nucleotide-exchange factor isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1) consists of 90 residues and differs from eukaryal EF-1s. The protein has been successfully crystallized using either microbatch-under-oil or vapour-diffusion methods. Crystals of native SsEF-1 diffract to 1.97 Å resolution and belong to space group P2 1 2 1 2, with unit-cell parameters a = 106.46, b = 54.87, c = 44.03 Å. Diffraction data have also been collected from a selenomethionine derivative of SsEF-1 at 1.83 Å resolution. Model building using the phases derived from the MAD experiment is in progress.