The plant-type ferredoxin/ferredoxin-NADP+ reductase (Fd/FNR) redox system found in parasites of the phylum Apicomplexa has been proposed as a target for novel drugs used against life-threatening diseases such as malaria and toxoplasmosis. Like many proteins from these protists, apicomplexan FNRs are characterized by the presence of unique peptide insertions of variable length and yet unknown function. Since three-dimensional data are not available for any of the parasite FNRs, we used limited proteolysis to carry out an extensive study of the conformation of Toxoplasma gondii FNR. This led to identification of 11 peptide bonds susceptible to the action of four different proteases. Cleavage sites are clustered in four regions of the enzyme, which include two of its three species-specific insertions. Such regions are thus predicted to form flexible surface loops. The protein substrate Fd protected FNR against cleavage both at its N-terminal peptide and at its largest sequence insertion (28 residues). Deletion by protein engineering of the species-specific subdomain containing the latter insertion resulted in an enzyme form that, although catalytically active, displayed a 10-fold decreased affinity for Fd. In contrast, removal of the first 15 residues of the enzyme unexpectedly enhanced its interaction with Fd. Thus, two flexible polypeptide regions of T. gondii FNR are involved in Fd interaction but have opposite roles in modulating the binding affinity for the protein ligand. In this respect, T. gondii FNR differs from plant FNRs, where the N-terminal peptide contributes to the stabilization of their complex with Fd.
|Titolo:||Roles of the species-specific subdomain and the N-terminal peptide of Toxoplasma gondii ferredoxin-NADP+ reductase in ferredoxin binding|
|Autori interni:||TEDESCHI, GABRIELLA|
PANDINI, VITTORIO ENRICO (Primo)
CAPRINI, GIANLUCA (Secondo)
ZANETTI, GIULIANA (Penultimo)
ALIVERTI, ALESSANDRO (Corresponding)
|Parole Chiave:||NADP+ reductase; plasmodium-falciparum; apicomplexan parasites; spinach ferredoxin; isoprenoid biosynthesis; Eschrichia-Coli; drug target; apicoplast; insertion; residues|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2006|
|Digital Object Identifier (DOI):||10.1021/bi052326w|
|Appare nelle tipologie:||01 - Articolo su periodico|
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