Simonetta Bartolucci

Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 °C; this is analogous to previous findings with mesophilic ADH at 25 °C ( ref. 5). Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units.

Protein disulfide bond formation is a rate limiting step in protein folding and is catalyzed by enzymes belonging to the protein disulfide oxidoreductase superfamily, including protein disulfide isomerase (PDI) in eucarya and DsbA in bacteria. The first high resolution X-ray crystal structure of a protein disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI. The protein consists of two homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The accessibilities of both active sites are rather different as are, very likely, their redox properties. The protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of disulfide bridges.

Characterization of the Sulfolobus host-SSV2 virus interaction.

The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report the findings of a comparative study of SSV2 physiology in the natural host, Sulfolobus islandicus REY15/4, versus the foreign host, Sulfolobus solfataricus, and provide evidence of differently regulated SSV2 life cycles in the two hosts. In fact, whereas a significant induction of SSV2 replication takes place during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus–host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural host, it was found that the SSV2 replication process was induced in the same way as in the natural host REY15/4.

Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70°C

Abstract The biocatalysts isolated from thermophilic microorganisms are the object of ever-growing scientific interest for (i) the comprehension of the molecular basis of their thermal tolerance, and (u) their use in different bio-industrial fields. Here we report the purification and characterization of an alcohol dehydrogenase (designated ADH-hT) from the novel strain LLD-R of Bacillus stearotbernsophilus which grows at 70°C. ADH-hT was obtained in pure form by anion exchange chromatography and two asinity chromatographies, with a final yield of about 30%. ADH-hT was found to be a tetramer of 37 kDa-subunits, and to have a pI of 4.9. ADH-hT displayed a broad substrate specificity; its activity was highest for aldehydes, and decreased progressively for alcohos and ketones. ADH-hT was endowed with catalytic activity and resistance in the presence of several denaturing agents (organic solvents, detergents, chaotropic agents). ADH-hT shared with ADH 1503 (the alcohol dehydrogenase from B. stearothermophilus strain NCA 1503 which grows at 55°C) the optimal temperature of 65°C, but it was more resistant than ADH 1503 towards beating. In conclusion, due to its stability and broad substrate specificity ADH-hT could be utilized in bio-industrial processes. Furthermore, we believe that ADH-hT could represent a good model system for studying ttie mechanism(s) which proteins exploit to gain heat resistance.

Structure and properties of a thermophilic and thermostable DNA polymerase isolated from Sulfolobus solfataricus

Summary A DNA-dependent DNA polymerase activity was purified to homogeneity from the archaebacterium Sulfolobus solfataricus , grown at 87°C and pH 3.5. This activity was the most abundant (80–85%) of two chromatographically distinguishable DNA polymerases. The enzyme purified about 1000-fold had a Mr of 210,000 ± 10,000 as determined by gel filtration. SDS gel electrophoresis revealed the presence of three peptides with a Mr of 116,000, 53,000 and 37,000, respectively, of which only the 116,000 subunit showed activity after elution from the gel and renaturation. However, by glycerol gradient centrifugation a Mr of 115,000 ± 5,000 was obtained. The DNA polymerase, assayed at 75°C and pH 6.8, required activated DNA and Mg ++ or Mn ++ for its activity and was thermophilic and thermostable. The temperature at which the activity was optimal depended on the type of DNA used as template-primer. It was concluded that the activity decreased at high temperature because of the melting of the template-primer, not as a result of enzyme inactivation. The DNA polymerase was also characterized with respect to its behaviour with inhibitors used to discriminate between enzymes isolated from prokaryotes or eukaryotes.

Identification and molecular characterization of an endoglucanase gene, celS, from the extremely thermophilic archaeon Sulfolobus solfataricus

Abstract. A genomic region upstream of the alcohol dehydrogenase (Ssadh) gene was cloned and sequenced from a library of Sulfolobus solfataricus MT4 strain. The isolated 4,040-bp DNA fragment revealed an open reading frame (celS), lying in the opposite direction to Ssadh, which showed significant similarity to endo-β-1,4-glucanases from Pyrococcus furiosus, Thermotoga maritima, and Thermotoga neapolitana. celS was shown to be a functional gene in vivo: a specific celS mRNA was detected by primer extension analysis showing a unique initiation transcription site coinciding with the ATG translation initiation codon. The specific gene product was detected as an extracellular cellulase after enzyme staining by carboxymethyl cellulose (CMC) SDS-PAGE, showing a molecular weight in agreement with that deduced from the open reading frame. Depending on growth conditions, different levels of cellulase activity and specific celS transcript were detected, revealing an inductive effect of CMC and suggesting a repressive role of glucose.

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (KM 0.3 µm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.

MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

A DNA binding protein, BldR, was identified in the crenarchaeon Sulfolobus solfataricus as a protein 5- to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the bldR gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the mar-like operon. The level of mar-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in E. coli, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and mar-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADH(Ss), stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.

Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress

Bcp2 was identified as a putative peroxiredoxin (Prx) in the genome database of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Its role in oxidative stress was investigated by transcriptional analysis of RNA isolated from cultures that had been stressed with various oxidant agents. Its specific involvement was confirmed by a considerable increase in the bcp2 transcript following induction with H2O2. The 5′ end of the transcript was mapped by primer extension analysis and the promoter region was characterized. bcp2 was cloned and expressed in Escherichia coli, the recombinant enzyme was purified and the predicted molecular mass was confirmed. Using dithiothreitol as an electron donor, this enzyme acts as a catalyst in H2O2 reduction and protects plasmid DNA from nicking by the metal‐catalysed oxidation system. Western blot analysis revealed that the Bpc2 expression was induced as a cellular adaptation in response to the addition of exogenous stressors. The results obtained indicate that Bcp2 plays an important role in the peroxide‐scavaging system in S. solfataricus. Mutagenesis studies have shown that the only cysteine, Cys49, present in the Bcp2 sequence, is involved in the catalysis. Lastly, the presence of this Cys in the sequence confirms that Bcp2 is the first archaeal 1‐Cysteine peroxiredoxin (1‐Cys Prx) so far identified.

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.