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Journal of Biochemistry Advance Access published online on January 29, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm051
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© 2007 The Japanese Biochemical Society

Determination of the Role of the Carboxyl-terminal Leucine-122 in FMN-binding Protein by Mutational and Structural Analysis

Masaya Kitamura1,*,{dagger}, Koji Terakawa1, Hideo Inoue1, Takuto Hayashida2, Kyoko Suto2, Yukio Morimoto2,3, Noritake Yasuoka2, Naoki Shibata2,3 and Yoshiki Higuchi2,3,{dagger}

1Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
2Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori Ako-gun, Hyogo 678-1297, Japan
3RIKEN Harima Institute/SPring-8, 1-1-1 Koto, Mikazuki-cho, Sayo-gun, Hyogo 679-5248, Japan

* Corresponding author: Masaya Kitamura, Ph. D., Address: Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan, Tel; +81-6-6605-3091, Fax; +81-6-6605-2782 E-mail: kitamura{at}bioa.eng.osaka-cu.ac.jp

Received December 25, 2006; Accepted January 3, 2007


  Abstract

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.

Key Words: crystal structure, dimer formation, dissociation constant, FMN-binding protein, redox potential

{dagger} The first and last authors contributed equally to the work.


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