Effects of temperature and SDS on the structure of beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus.
Ontology highlight
ABSTRACT:
SUBMITTER: D'auria S
PROVIDER: S-EPMC1218389 | biostudies-other | 1997 May
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of 'chimeric' proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.
| S-EPMC1976387 | biostudies-literature
Project description:The existence of a global gene regulatory system in the hyperthermophilic archaeon Sulfolobus solfataricus is described. The system is responsive to carbon source quality and acts at the level of transcription to coordinate synthesis of three physically unlinked glycosyl hydrolases implicated in carbohydrate utilization. The specific activities of three enzymes, an alpha-glucosidase (malA), a beta-glycosidase (lacS), and an alpha-amylase, were reduced 4-, 20-, and 10-fold, respectively, in response to the addition of supplementary carbon sources to a minimal sucrose medium. Western blot analysis using anti-alpha-glucosidase and anti-beta-glycosidase antibodies indicated that reduced enzyme activities resulted exclusively from decreased enzyme levels. Northern blot analysis of malA and lacS mRNAs revealed that changes in enzyme abundance arose primarily from reductions in transcript concentrations. Culture conditions precipitating rapid changes in lacS gene expression were established to determine the response time of the regulatory system in vivo. Full induction occurred within a single generation whereas full repression occurred more slowly, requiring nearly 38 generations. Since lacS mRNA abundance changed much more rapidly in response to a nutrient down shift than to a nutrient up shift, transcript synthesis rather than degradation likely plays a role in the regulatory response.
| S-EPMC93880 | biostudies-literature
Project description:The stability and activity of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus were studied as a function of pH and temperature. In this paper we focus on three points: (1) the long-term stability of the protein to irreversible denaturation at high temperature; (2) the short-term stability of the protein to reversible temperature-driven unfolding; and (3) the dependence of its activity on temperature. Results can be summarized as follows: (a) the same first-order kinetic constant (0.020+/-0.003 min-1) was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 degrees C; (b) short-term stability experiments revealed different behaviour in two different pH ranges (6.5-8.0, 8.5-9.5), suggesting that the melting temperature is higher at alkaline than at neutral pH; (c) the dependence of activity on temperature was investigated at pH 7.0 and 9.0, and a discontinuity was observed in the Arrhenius plot of kcat values at pH 9.0. We also investigated the stability in the presence of guanidinium chloride at 20 degrees C either at pH 7.0 or at pH 9.0, and we present data that indicate that the unfolding mechanism closely approaches a two-state model at pH 7.0 and a more complex mechanism at pH 9.0. Satisfactory fitting of the equilibrium unfolding transition obtained by fluorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 degrees C the folded conformation is more stable than the unfolded conformation by (14. 7+/-1.2) kJ/mol at pH 7.0 and by (25.5+/-1.8) kJ/mol at pH 9.0.
| S-EPMC1218304 | biostudies-other
Project description:The thermoacidophilic archaeon Sulfolobus solfataricus P2 encodes three hypothetic endo-beta-glucanases, SSO1354, SSO1949 and SSO2534. We cloned and expressed the gene sso1949 encoding the 334 amino acids containing protein SSO1949, which can be classified as a member of glycoside hydrolase family 12. The purified recombinant enzyme hydrolyses carboxymethylcellulose as well as cello-oligomers, with cellobiose and cellotriose as main reaction products. By following the hydrolysis of a fluorescently labelled cellohexaoside under a wide variety of conditions, we show that SSO1949 is a unique extremophilic enzyme. This archaeal enzyme has a pH optimum of approx. pH 1.8 and a temperature optimum of approx. 80 degrees C. Furthermore, the enzyme is thermostable, with a half-life of approx. 8 h at 80 degrees C and pH 1.8. The thermostability is strongly pH-dependent. At neutral pH, the thermal inactivation rate is nearly two orders of magnitude higher than at pH 1.8. Homology modelling suggests that the catalytic domain of SSO1949 has a similar fold to other mesophilic, acidophilic and neutral cellulases. The presence of a signal peptide indicates that SSO1949 is a secreted protein, which enables S. solfataricus to use cellulose as an external carbon source. It appears that SSO1949 is perfectly adapted to the extreme environment in solfataric pools. A cellulolytic enzyme with such a combination of stability and activity at high temperatures and low pH has not been described so far and could be a valuable tool for the large-scale hydrolysis of cellulose under acidic conditions.
| S-EPMC1134732 | biostudies-literature
Project description:Beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus is a homotetramer with a higher number of ion pairs compared with mesophilic glycoside hydrolases. The ion pairs are arranged in large networks located mainly at the tetrameric interface of the molecule. In the present study, the structure and thermal stability of the wild-type beta-glycosidase and of three mutants in residues R488 and H489 involved in the C-terminal ionic network were examined by FTIR (Fourier-transform IR) spectroscopy. The FTIR data revealed small differences in the secondary structure of the proteins and showed a lower thermostability of the mutant proteins with respect to the wild-type. Generalized 2D-IR (two-dimensional IR correlation spectroscopy) at different temperatures showed different sequences of thermal unfolding events in the mutants with respect to the wild-type, indicating that punctual mutations affect the unfolding and aggregation process of the protein. A detailed 2D-IR analysis of synchronous maps of the proteins allowed us to identify the temperatures at which the ionic network that stabilizes the quaternary structure of the native and mutant enzymes at the C-terminal breaks down. This evidence gives support to the current theories on the mechanism of ion-pair stabilization in proteins from hyperthermophilic organisms.
| S-EPMC1134089 | biostudies-literature
Project description:The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (?-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ?50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.
| S-EPMC4005642 | biostudies-literature
Project description:When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [gamma-(32)P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of approximately 46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co(2+) or Mn(2+). The recombinant protein underwent autophosphorylation when incubated with either [gamma-(32)P]ATP or [gamma-(32)P]GTP. The site of phosphorylation was identified as Ser(59), which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the (32)P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of (32)P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.
| S-EPMC151489 | biostudies-literature
Project description:In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.
| S-EPMC2168930 | biostudies-literature
Project description:Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cycle arrest and modulation of gene expression. Genomic sequencing has revealed the presence of archaeal genes homologous to components of the eucaryal nucleotide excision repair (NER) pathway, which is involved in the repair of ultraviolet (UV) light-induced DNA damage. However, the events involved in the cell response to UV irradiation and their regulation have not been studied in Archaea. We show here that UV radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) in the hyperthermophilic archaeon Sulfolobus solfataricus, and that these lesions are efficiently repaired in vivo in the dark, suggesting that a NER pathway is active. DNA damage is a signal for concomitant growth arrest and transcriptional induction of the NER genes XPF, XPG and XPB. The cell response to UV irradiation includes transcriptional regulation of genes encoding two DNA binding proteins involved in chromosome dynamics. Moreover, several of these genes are also strongly induced by the intercalating agent actinomycin D. Thus, response to DNA damage in S.solfataricus has features essentially conserved in all three domains of life.
| S-EPMC275473 | biostudies-literature
Project description:Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricusIMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.
| S-EPMC6328758 | biostudies-literature