Cold-adapted monomeric isocitrate dehydrogenase of a psychrophilic bacterium,
Colwellia maris, (
CmIDH) showed a high degree of amino acid sequential identity (69.5%) to a mesophilic nitrogen-fixing bacterium,
Azotobacter vinelandii, (
AvIDH). In this study, three Ala residues of
CmIDH and the corresponding Pro residues of
AvIDH were exchanged by site-directed mutagenesis, and several properties of single, double, and triple mutants of the two enzymes were investigated. The mutated
CmIDHs, which replaced Ala719 with Pro, showed increased activity and elevation of the optimum temperature and thermostability for activity. In contrast, mutants of
AvIDH, in which Pro717 was replaced by Ala, decreased the thermostability for activity. These results indicate that Ala719 of
CmIDH and Pro717 of
AvIDH are involved in thermostability. On the other hand, mutated
CmIDH, in which Ala710 was replaced by Pro, and the corresponding
AvIDH mutant, which replaced Pro708 with Ala, showed higher and lower specific activity than the corresponding wild-type enzymes, suggesting that Pro708 of
AvIDH is involved in its high catalytic ability. Furthermore, the exchange mutations between Ala740 in
CmIDH and the corresponding Pro738 in
AvIDH resulted in decreased and increased thermostability for
CmIDH and
AvIDH activity respectively, suggesting that the native Ala740 and Pro738 residues make the enzymes thermostable and thermolabile.
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