Functional and structural characterization of protein disulfide oxidoreductase from Thermus thermophilus HB27.
The paper reports the characterization of a protein disulfide oxidoreductase (PDO) from the thermophilic Gram negative bacterium Thermus thermophilus HB27, identified as TTC0486 by genome analysis and named TtPDO. PDO members are involved in the oxidative folding, redox balance and detoxification of peroxides in thermophilic prokaryotes. Ttpdo was cloned and expressed in E. coli and the recombinant purified protein was assayed for the dithiol-reductase activity using insulin as substrate and compared with other PDOs characterized so far. In the thermophilic archaeon Sulfolobus solfataricus PDOs work as thiol-reductases constituting a peculiar redox couple with Thioredoxin reductase (SsTr). To get insight into the role of TtPDO, a hybrid redox couple with SsTr, homologous to putative Trs of T. thermophilus, was assayed. The results showed that SsTr was able to reduce TtPDO in a concentration dependent manner with a calculated K M of 34.72 μM, suggesting the existence of a new redox system also in thermophilic bacteria. In addition, structural characterization of TtPDO by light scattering and circular dichroism revealed the monomeric structure and the high thermostability of the protein. The analysis of the genomic environment suggested a possible clustering of Ttpdo with TTC0487 and TTC0488 (tlpA). Accordingly, transcriptional analysis showed that Ttpdo is transcribed as polycistronic messenger. Primer extension analysis allowed the determination of its 5'end and the identification of the promoter region.
Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Naples, Italy.
Emilia
E
Pedone
Gabriella
G
Fiorentino
Luciano
L
Pirone
Patrizia
P
Contursi
Simonetta
S
Bartolucci
Danila
D
Limauro
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D011490:Protein Disulfide Reductase (Glutathione) / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*
D001426:Bacterial Proteins; D011490:Protein Disulfide Reductase (Glutathione)