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.
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.
Thomsen-Zieger
Nadine
N
FB Biologie/Parasitologie, Philipps-Universität Marburg, Karl-von-Frisch-Str., D-35032 Marburg, Germany.
Pandini
Vittorio
V
Caprini
Gianluca
G
Aliverti
Alessandro
A
Cramer
Jörg
J
Selzer
Paul M
PM
Zanetti
Giuliana
G
Seeber
Frank
F
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*
D005288:Ferredoxins; D015800:Protozoan Proteins; D005287:Ferredoxin-NADP Reductase