Toxoplasma gondii:: analysis of the active site insertion of its ferredoxin-NADP+-reductase by peptide-specific antibodies and homology-based modeling

Apicomplexan parasites possess an apicoplast-localized redox system consisting of a plant-type ferredoxin-NADP(+)-reductase (FNR) and its redox partner ferredoxin, a small [2Fe-2S] protein. We show here that several apicomplexan FNRs contain unique amino acid insertions of various lengths which are located in close proximity to the enzymatically important FAD and ferredoxin-binding sites of these proteins. Using the insertion of the Toxoplasma gondii reductase as an example we raised epitope-specific antibodies against an I I amino acids long peptide predicted to be surface-exposed within this insertion. This peptide was found to be immunogenic when presented to the immune system as part of a carrier protein, but also in its natural structural context in the whole recombinant protein, implying that the epitope is surface-exposed. Three-dimensional modeling of T gondii FNR based on the known 3D-structure of maize root FNR predicts that the overall structure of plant and apicomplexan FNRs are very similar and that the I I amino acids are part of an alpha-helix, looping out of the molecule. Collectively, these data suggest that the insertion in T gondii FNR does not affect the overall structure of the protein but may have an effect on the binding dynamics of FAD, NADP(+), and/or ferredoxin to FNR. (C) 2003 Elsevier Science (USA). All rights reserved.