Effects of the substituted amino acid residues on the thermal properties of monomeric isocitrate dehydrogenases from a psychrophilic bacterium, Psychromonas marina , and a mesophilic bacterium, Azotobacter vinelandii

A cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina ( Pm IDH), showed a high degree of amino acid sequential identity (64%) to a mesophilic one from a mesophilic bacterium, Azotobacter vinelandii ( Av IDH). In this study, eight corresponding amino acid residues were substituted between them by site-directed mutagenesis, and several thermal properties of the mutated IDHs were examined. In the Pm IDH mutants, Pm L735F, substituted Leu735 of Pm IDH by the corresponding Phe of Av IDH, showed higher specific activity and thermostability of activity than wild-type Pm IDH, while the H600Y and N741P mutations of Pm IDH resulted in decreased specific activity and thermostability of activity. On the other hand, among the Av IDH mutants, Av P718T showed lower optimum temperature and thermostability of activity than wild-type Av IDH. In Pm IDH variously combined the H600Y, L735F and N741P mutations, Pm H600YL735F, including the H600Y and L735F mutations, showed higher specific activity than Pm H600Y and similar optimum temperature and thermostability of activity to Pm H600Y. Furthermore, Pm L735FN741P exhibited higher specific activity and thermostability of activity than Pm N741P. These results indicated that the effects of the three mutations of Pm IDH are additive on the specific activity of both Pm H600YL735F and Pm L735FN741P and on thermostability of Pm L735FN741P.