title: Analysis of amino acid residues involved in cold activity of monomeric isocitrate dehydrogenase from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea.
abstract: Monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH-II and CpIDH-M, respectively) are cold-adapted enzymes and show a high degree of amino acid sequential identity to each other (77%). However, maximum activity of CpIDH-M at optimum temperature is much less than that of CmIDH-II. In the C-terminal region 3 of these enzymes, which was suggested from previous study to be responsible for their distinct catalytic ability, several sequential differences of amino acid residue are present. Among them, ten amino acid residues were exchanged between them by site-directed mutagenesis and several properties of the mutated enzymes were examined in this study. The mutated enzymes of CmIDH-II substituted its Gln671, Leu724 and Phe735 residues with the corresponding residues of CpIDH-M (termed Q671K, L724Q and F735L, respectively) showed lower specific activity and thermostability for activity than the wild-type enzyme. Furthermore, the decreased specific activity was also observed in L693F. In contrast, the corresponding mutants of CpIDH-M, F693L, Q724L and L735F, showed the increased specific activity and thermostability for activity. The catalytic efficiency (k(cat)/K(m)) values of these mutated CmIDH-II and CpIDH-M were lower and higher than those of their wild-type IDHs, respectively. These results suggest that the Gln671, Leu693, Leu724 and Phe735 residues of CmIDH-II are important for exerting its high catalytic ability.
authors.affiliation: Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku, Sapporo 060-0810, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Yasuda
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takada
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D003080:Cold Temperature; D004795:Enzyme Stability / Q000235:genetics; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
chemical_list: D007521:Isocitrate Dehydrogenase
keywords: Cold-adapted enzyme; Colwellia maris; Colwellia psychrerythraea; Isocitrate dehydrogenase; Site-directed mutagenesis