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Mosè Rossi

Publications and source records attributed to Mosè Rossi.

At least 73 records · Page 4Linked to original sources
PubMed2004

Functional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a member of a novel protein family related to protein disulfide-isomerase.

Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol-disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 degrees C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family.

Adenosine Triphosphate↗
PubMed2003

Crystal structure of a ternary complex of the alcohol dehydrogenase from Sulfolobus solfataricus.

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.

Alcohol Dehydrogenase↗
PubMed2003

Glycosynthase-catalysed syntheses at pH below neutrality.

The use of the new glycosynthase Ta-beta-GlyE386G and of the already known Ss-beta-GlyE387G for the synthesis of interesting 4-methylumbelliferyl disaccharides and for the galactosylation of alpha- and beta-xylosides of 4-penten-1-ol is reported. The results show satisfactory yields of reaction in presence of low excesses of acceptors and demonstrated that the high activity of these enzymes at pH below neutrality is applicable in the transfer of glucose as well as of galactose from the preferred 2-NP-based donors.

Amino Acid Substitution↗
PubMed2003

A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis.

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.

Alcohols↗
PubMed2003

Transcriptional response to DNA damage in the archaeon Sulfolobus solfataricus.

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.

Archaeal Proteins↗
PubMed2003

Crystallization and preliminary X-ray diffraction studies of Aes acetyl-esterase from Escherichia coli.

The acetyl-esterase Aes from Escherichia coli, which belongs to the HSL group of the esterase/lipase superfamily, has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as a precipitant and magnesium chloride as an additive. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 110.0, b = 190.6, c = 218.6 A. A complete data set has been collected to 2.5 A resolution at the Elettra synchrotron source, Trieste using a single frozen crystal. Packing density considerations agree with 10-16 monomers in the asymmetric unit, with a corresponding solvent content of 61-38%.

Acetylesterase↗
PubMed2003

Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solfataricus.

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.

Adenosine Triphosphate↗
PubMed2003

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Sulfolobus solfataricus via chemical rescue of an inactive mutant.

We have recently reported that a functional alpha-L-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.

Amino Acid Sequence↗
PubMed2003

Structural and thermal stability analysis of Escherichia coli and Alicyclobacillus acidocaldarius thioredoxin revealed a molten globule-like state in thermal denaturation pathway of the proteins: an infrared spectroscopic study.

The structure of thioredoxin from Alicyclobacillus acidocaldarius (previously named Bacillus acidocaldarius ) (BacTrx) and from Escherichia coli ( E. coli Trx) was studied by Fourier-transform IR spectroscopy. Two mutants of BacTrx [Lys(18)-->Gly (K18G) and Arg(82)-->Glu (R82E)] were also analysed. The data revealed similar secondary structures in all proteins, but BacTrx and its mutants showed a more compact structure than E. coli Trx. In BacTrx and its mutants, the compactness was p(2)H-dependent. All proteins revealed the existence of a molten globule-like state. At p(2)H 5.8, the temperature at which this state was detected was higher in BacTrx and decreased in the different proteins in the following order: BacTrx>R82E>K18G> E. coli Trx. At neutral or basic p(2)H, the molten globule-like state was detected at the same temperature in both BacTrx and R82E, whereas it was found at the same temperature in all p(2)Hs tested for E. coli Trx. The thermal stability of the proteins was in the following order at all p(2)Hs tested: BacTrx>R82E>K18G> E. coli Trx, and was lower for each protein at p(2)H 8.4 than at neutral or acidic p(2)Hs. The formation of protein aggregates, brought about by thermal denaturation, were observed for BacTrx and K18G at all p(2)Hs tested, whereas they were present in R82E and E. coli Trx samples only at p(2)H 5.8. The results indicated that a single mutation might affect the structural properties of a protein, including its propensity to aggregate at high temperatures. The data also indicated a possible application of Fourier-transform IR spectroscopy for assessing molten globule-like states in small proteins.

Enzyme Stability↗
PubMed2003

Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH.

We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus solfataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the beta-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.

Amino Acid Sequence↗
PubMed2003

Effect of trifluoroethanol on the conformational stability of a hyperthermophilic esterase: a CD study.

The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of alpha-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v(-1)), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 degrees C for native AFEST, but becomes 85 degrees C at 1.4 M TFE (10% v v(-1)), and 56 degrees C at 2.8 M TFE (20% v v(-1)). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.

Animals↗
PubMed2003

Structural study of a single-point mutant of Sulfolobus solfataricus alcohol dehydrogenase with enhanced activity.

Alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) is the only enzyme from Archaea among the structurally studied members of the medium-chain ADH family described so far. Here, we present the three-dimensional structure of the apo form of the mutant N249Y which exhibits increased catalytic activity when compared to the wild-type enzyme. The substitution, located in the coenzyme binding domain, decreases the affinity for NAD(H) cofactor. The rearrangement of segments 248-250 and 270-275, induced by the mutation, suggests an explanation for the lower coenzyme affinity. This study also highlights the role in SsADH catalysis of the flexible loops located at the interface between the catalytic and the coenzyme domains.

Alcohol Dehydrogenase↗
PubMed2003

Identification of an archaeal alpha-L-fucosidase encoded by an interrupted gene. Production of a functional enzyme by mutations mimicking programmed -1 frameshifting.

The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.

Archaea↗
PubMed2003

High-level expression of Aliciclobacillus acidocaldarius thioredoxin in Pichia pastoris and Bacillus subtilis.

Thioredoxins are ubiquitous proteins which catalyze the reduction of disulfide bridges on target proteins and are involved in many cellular reactions. In a previous work, a thioredoxin from the thermophilic organism Aliciclobacillus acidocaldarius (Alitrx) was purified, characterized, and its gene expressed in Escherichia coli. In order to produce larger quantities of Alitrx, the protein has been expressed in the methylotrophic yeast Pichia pastoris and in the gram positive bacteria Bacillus subtilis. The growth conditions of strains showing high-level expression of Alitrx were optimized for both systems in shake-flask cultures. Active proteins were secreted in the culture media at a level of approximately 0.9 and 0.5 g/l, respectively, for P. pastoris and B. subtilis. The proteins were purified almost to homogeneity by a thermal precipitation procedure, with a 90-fold and 50-fold higher total yield with respect to that obtained with the same protein expressed in E. coli. The results indicate that either of these two systems could be utilized as a host for large-scale production of recombinant Alitrx.

Bacillus subtilis↗
PubMed2003

Transcriptional regulation of the gene encoding an alcohol dehydrogenase in the archaeon Sulfolobus solfataricus involves multiple factors and control elements.

A transcriptionally active region has been identified in the 5' flanking region of the alcohol dehydrogenase gene of the crenarchaeon Sulfolobus solfataricus through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances. Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors. When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed. To understand the molecular mechanisms underlying the substrate-induced expression of the adh gene, the promoter was analyzed for the presence of cis-acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde. Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences. A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL cis-acting element was also identified. This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of approximately 16 kDa. The correlation between S. solfataricus adh gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes.

5' Flanking Region↗
PubMed2003

Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus.

LacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of approximately 13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside).

Base Sequence↗
PubMed2002

An intracellular protease of the crenarchaeon Sulfolobus solfataricus, which has sequence similarity to eukaryotic peptidases of the CD clan.

We purified from crude extracts of the hyperthermophilic crenarchaeon Sulfolobus solfataricus a protease that is able to hydrolyse proteins with a pH optimum of 7.5 and a temperature optimum of 70 degrees C. Assays in the presence of classical protease inhibitors showed that the hydrolytic activity is sensitive to thiol-blocking reagents. Fluorescence assays using synthetic peptides demonstrated that the protease has a preference for cleaving glutamic acid residues. The first 12 residues of the protease match the N-terminus residues of a hypothetical protein in the S. solfataricus genome of 95 amino acids in length and calculated molecular mass of 11072 Da. The whole sequence of the protease is not related to any known protein, but it bears a segment which is highly similar to one containing the active cysteine residue in eukaryotic peptidases known as legumains. This is the first protease isolated from S. solfataricus capable of degrading native proteins effectively. Our results add to the knowledge of the intracellular proteolytic machine in hyperthermophilic micro-organisms.

Amino Acid Sequence↗