Electron transfer between plant-type [2Fe-2S] ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) depends on the physical interaction between both proteins. We have applied a random mutagenesis approach with subsequent in vivo selection using the yeast two-hybrid system to obtain mutants of Toxoplasma gondii FNR with higher affinity for Fd. One mutant showed a 10-fold enhanced binding using affinity chromatography on immobilized Fd. A single serine-to-arginine exchange in the active site was responsible for its increased affinity. The mutant reductase was also enzymatically inactive. Homology modeling of the mutant FNR-Fd complex predicts substantial alterations of protein-FAD interactions in the active site of the enzyme with subsequent structural changes. Collectively, for the first time a point mutation in this important class of enzymes is described which leads to greatly enhanced affinity for its protein ligand.
Titolo: | A single in vivo-selected point mutation in the active center of Toxoplasma gondii ferredoxin-NADP+ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin |
Autori: | PANDINI, VITTORIO ENRICO (Secondo) ZANETTI, GIULIANA (Penultimo) |
Parole Chiave: | Affinity mutant; Apicomplexa; Apicoplast; Ferredoxin NADP+ reductase; Two-hybrid system |
Settore Scientifico Disciplinare: | Settore BIO/10 - Biochimica |
Data di pubblicazione: | 22-ott-2004 |
Rivista: | |
Tipologia: | Article (author) |
Digital Object Identifier (DOI): | 10.1016/j.febslet.2004.09.042 |
Appare nelle tipologie: | 01 - Articolo su periodico |
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