| Autor: |
Pedone E., D’Alterio R., D’ambrosio K., De Simone G., LIMAURO, DANILA, ROSSI, MOSE', BARTOLUCCI, SIMONETTA |
| Přispěvatelé: |
Pedone, E., Limauro, Danila, D’Alterio, R., D’Ambrosio, K., De Simone, G., Rossi, Mose', Bartolucci, Simonetta |
| Jazyk: |
angličtina |
| Rok vydání: |
2006 |
| Popis: |
Genomic evidence has recently implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from thermophilic organisms, contrary to the conventional view that structural disulfide bonds are exclusively of extracellular proteins. Both computational and structural data have been presented to explore the abundance of disulfide bonds as a protein stabilization method across many thermophilic prokaryotes [1]. Interestingly, only a distinct subset of thermophiles exhibit this property and all of them have a protein disulphide oxidoreductase (PDO), belonging to PDI-like family, indicated as a potential player in thermophilic intracellular disulfide bond formation [2,3]. Members of this family are characterized by a molecular mass of about 26 kDa and by two Trx folds, each comprising a CXXC active site motif. In order to provide insights into the function, structural diversity and evolution of PDOs, this work focuses on the structural and functional characterization of three members of this family, which were isolated from the thermophilic bacterium Aquifex aeolicus (AaPDO) [4] and from the archaea Aeropyrum pernix (ApPDO) [5] and Sulfolobus solfataricus (SsPDO), in comparison with the already characterized eukaryotic PDI and the PDO from the archaeon Pyrococcus furiosus (PfPDO) [6]. The proteins were cloned, expressed and purified. Reductive, oxidative and isomerase assays were performed on all the PDOs. Site-directed mutagenesis experiments on AaPDO were used to assess the involvement of the two active sites in catalytic activities, while theoretical calculations of the cysteine pKa values of ApPDO were performed and compared with values of the other PDOs. In addition, S. solfataricus has been chosen as a model system to study the role of SsPDO in the formation of disulfides and in the involvement in a thioredoxin-like system with a thioredoxin reductase, SsTR. Functional studies have revealed the capability of these enzyme in reducing, oxidizing and isomerising disulfide bridges demonstrating that they are members of the PDI-like family. The resolution of the structures of AaPDO and ApPDO showed a correlation with the already known PDI structure. In addition, peculiar structural characteristics, site-directed mutagenesis experiments and computational studies have suggested that their two active sites have equivalent functional properties. SsPDO resulted to constitute a thioredoxin system with SsTR. The reported data indicate that SsPDO, being involved in a thioredoxin-like system, plays a central role in the biochemistry of cytoplasmic disulfide bonds in the thermophilic S. solfataricus. In addition, as the different three-dimensional features of these biological systems strongly suggest significantly different mechanisms of action, further experimental studies of these enzyme families will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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