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.
Pedone
Emilia
E
Istituto di Biostrutture e Bioimmagini, C.N.R., Napoli, Italy.
Ren
Bin
B
Ladenstein
Rudolf
R
Rossi
Mosè
M
Bartolucci
Simonetta
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D012260:Ribonucleases / Q000378:metabolism
D007328:Insulin; D000255:Adenosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; C113416:protein disulfide oxidoreductase, P. furiosus; D012260:Ribonucleases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine