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
Articolo
Data di Pubblicazione:
2004
Citazione:
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 / N. Thomsen-Zieger, V. Pandini, G. Caprini, A. Aliverti, J. Cramer, P. M. Selzer, G. Zanetti, F. Seeber. - In: FEBS LETTERS. - ISSN 0014-5793. - 576:3(2004 Oct 22), pp. 375-380.
Abstract:
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.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
Affinity mutant; Apicomplexa; Apicoplast; Ferredoxin NADP+ reductase; Two-hybrid system
Elenco autori:
N. Thomsen-Zieger, V. Pandini, G. Caprini, A. Aliverti, J. Cramer, P. M. Selzer, G. Zanetti, F. Seeber
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