A cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina (PmIDH), showed a high degree of amino acid sequential identity (64%) to a mesophilic one from a mesophilic bacterium, Azotobacter vinelandii (AvIDH). 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 PmIDH mutants, PmL735F, substituted Leu735 of PmIDH by the corresponding Phe of AvIDH, showed higher specific activity and thermostability of activity than wild-type PmIDH, while the H600Y and N741P mutations of PmIDH resulted in decreased specific activity and thermostability of activity. On the other hand, among the AvIDH mutants, AvP718T showed lower optimum temperature and thermostability of activity than wild-type AvIDH. In PmIDH variously combined the H600Y, L735F and N741P mutations, PmH600YL735F, including the H600Y and L735F mutations, showed higher specific activity than PmH600Y and similar optimum temperature and thermostability of activity to PmH600Y. Furthermore, PmL735FN741P exhibited higher specific activity and thermostability of activity than PmN741P. These results indicated that the effects of the three mutations of PmIDH are additive on the specific activity of both PmH600YL735F and PmL735FN741P and on thermostability of PmL735FN741P.

中文翻译：

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取代的氨基酸残基对嗜冷细菌Psychromonas marina和嗜温细菌Azotobacter vinelandii的单体异柠檬酸脱氢酶热性质的影响。

一种来自嗜冷细菌Psychromonas marina（Pm IDH）的冷适应单体异柠檬酸脱氢酶，与来自嗜温细菌Azotobacter vinelandii（Av IDH）的嗜温细菌具有高度的氨基酸序列同一性（64％）。在这项研究中，通过定点诱变将八个对应的氨基酸残基替换为它们之间，并检测了突变的IDH的几种热性质。在Pm IDH突变体中，Pm L735F（被相应的Av IDH的Phe取代了Pm IDH的Leu735）比野生型Pm具有更高的比活性和活性的热稳定性IDH，而Pm IDH的H600Y和N741P突变导致比活性降低和活性热稳定性降低。另一方面，在Av IDH突变体中，Av P718T的最佳温度和活性的热稳定性低于野生型Av IDH。在PM IDH不同地组合的H600Y，L735F和N741P突变，PM H600YL735F，包括H600Y和L735F突变，显示出比更高的比活性PM H600Y和类似的最适温度和活性的热稳定性，以PM H600Y。此外，Pm L735FN741P的比活性和活性的热稳定性均高于Pm N741P。这些结果表明，三个突变的影响PM IDH是双方的具体活性添加剂PM H600YL735F和PM L735FN741P和热稳定性PM L735FN741P。