Watanabe Keiko, Yamagishi Akihiko
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
FEBS Lett. 2006 Jul 10;580(16):3867-71. doi: 10.1016/j.febslet.2006.06.012. Epub 2006 Jun 16.
Previously, we showed that mutants of Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70 degrees C when compared with the wild-type IPMDH.
此前，我们发现嗜热栖热菌3-异丙基苹果酸脱氢酶（IPMDH）的突变体，每个突变体都含有一个预测存在于假定共同祖先蛋白中的残基（祖先残基），其热稳定性通常比当代野生型酶更高。在本研究中，分析了多个祖先残基的综合作用。构建了两个含有多个突变的突变体Sup3mut（Val181Thr/Pro324Thr/Ala335Glu）和Sup4mut（Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu），它们表现出比野生型和单点突变IPMDH更高的热稳定性。与野生型IPMDH相比，大多数突变体在70摄氏度时具有相似或更高的催化效率。
