Determination of the role of the Carboxyl-terminal leucine-122 in FMN-binding protein by mutational and structural analysis.
Mutants of flavin mononucleotide-binding protein (FMN-bp) were made by site-directed mutagenesis to investigate the role of carboxyl-terminal Leu122 of the pairing subunit in controlling redox potentials, binding the prosthetic group, and forming the tertiary and quaternary structure. We compared the oxidation-reduction potentials, FMN-binding properties, and higher structures of wild-type FMN-bp and four mutant proteins (L122Y, L122E, L122K and L122-deleted). We found that the redox potentials were affected by mutations. Also, the affinities of L122E, L122K and L122 deletion mutant apoproteins for FMN were lower than for the wild-type apoprotein, whereas the affinity of L122Y for FMN was increased. Analytical ultracentrifugation showed that the dissociation constants for dimerization of L122E and L122K were larger than for wild-type FMN-bp, whereas the dissociation constants for L122Y and the deletion mutant were lower than for the wild type. Finally, we determined the higher structures of L122Y, L122E and L122K mutants by X-ray crystallography. Our results show that the mutation of Leu122 in FMN-bp changes midpoint potentials, dissociation constants for FMN, and dimer formation, indicating that this residue is important in the pairing subunit.
Kitamura
Masaya
M
Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto, Osaka, Japan. kitamura@bioa.eng.osaka-cu.ac.jp
Terakawa
Koji
K
Inoue
Hideo
H
Hayashida
Takuto
T
Suto
Kyoko
K
Morimoto
Yukio
Y
Yasuoka
Noritake
N
Shibata
Naoki
N
Higuchi
Yoshiki
Y
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D016969:Desulfovibrio vulgaris / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D001426:Bacterial Proteins; C086046:FMN-binding protein, Desulfovibrio vulgaris; D005420:Flavoproteins; D005486:Flavin Mononucleotide; D007930:Leucine