title: 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.
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.
authors.affiliation: FB Biologie/Parasitologie, Philipps-Universität Marburg, Karl-von-Frisch-Str., D-35032 Marburg, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Thomsen-Zieger
authors.forename: Vittorio
authors.initials: V
authors.lastname: Pandini
authors.forename: Gianluca
authors.initials: G
authors.lastname: Caprini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aliverti
authors.forename: Jörg
authors.initials: J
authors.lastname: Cramer
authors.forename: Paul M
authors.initials: PM
authors.lastname: Selzer
authors.forename: Giuliana
authors.initials: G
authors.lastname: Zanetti
authors.forename: Frank
authors.initials: F
authors.lastname: Seeber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000201:enzymology*
chemical_list: D005288:Ferredoxins; D015800:Protozoan Proteins; D005287:Ferredoxin-NADP Reductase