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 |
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Authors: | Thomsen-Zieger Nadine Pandini Vittorio Caprini Gianluca Aliverti Alessandro Cramer Jörg Selzer Paul M Zanetti Giuliana Seeber Frank |
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Institution: | FB Biologie/Parasitologie, Philipps-Universit?t Marburg, Karl-von-Frisch-Str., D-35032 Marburg, Germany. |
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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. |
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Keywords: | HA hemaglutinin epitope tag Fd ferredoxin FNR ferredoxin-NADP+ oxidoreductase mutTgFNR double-mutant of TgFNR PfFd P falciparum Fd sl spinach leaf wt wild-type TgFd T gondii Fd TgFNR T gondii FNR |
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