title: Effects of the combined substitutions of amino acid residues on thermal properties of cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria.
abstract: In the two cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH and CpIDH, respectively), the combined substitutions of amino acid residues between the Leu693, Leu724 and Phe735 residues of CmIDH and the corresponding Phe693, Gln724 and Leu735 residues of CpIDH were introduced by site-directed mutagenesis. A double mutant of CmIDH substituted its Leu724 and Phe735 residues by the corresponding ones of CpIDH, CmL724Q/F735L, and the triple mutant of CpIDH, CpF693L/Q724L/L735F, showed the most decrease and increase of activity, respectively, of each wild-type and its all mutated enzymes. In the case of CmIDH, the substitutions of these three amino acid residues resulted in the decrease of catalytic activity and thermostability for activity, but the combined substitutions of amino acid residues did not necessarily exert additive effects on these properties. On the other hand, similar substitutions in CpIDH had quite opposite effects to CmIDH, and the effects of the combined substitutions were additive. All multiple mutants of CmIDH and CpIDH showed lower and higher catalytic efficiency (k(cat)/K(m)) values than the respective wild-type enzymes. Single and multiple mutations of the substituted amino acid residues in the CmIDH and CpIDH led to the increase and decrease of sensitivity to tryptic digestion, indicating that the stability of protein structure was decreased and increased by the mutations, respectively.
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: Miyuki
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takada
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004795:Enzyme Stability; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D007521:Isocitrate Dehydrogenase