Stabilization mechanism of proteins from hyperthermophilic archaeon

为了了解生长在95 ℃以上的极端嗜热古菌的蛋白质适应异常高温的机制，我们从Pyrococcus kodakaraensis KODI（Pk）中克隆了编码甘油激酶（GK）和核糖核酸酶HII（RNase HII）的基因。将这些基因在大肠杆菌中过表达，并将重组酶纯化至均一。热处理被证明是非常有效的纯化这些酶，因为大多数来自大肠杆菌的蛋白质在热处理时由于热变性而沉淀，并通过离心除去。Pk-GK和Pk-RNase HII由497和288个氨基酸残基组成，分别以二聚体和单体形式存在。这些酶显示出不寻常的酶特性，如对热失活的高稳定性和广泛的金属离子特异性。与中温来源的酶的氨基酸组成比较表明，Pk-GK和Pk-RNase HII中的带电荷氨基酸残基含量分别比E. coliGK和E. coliRNase HII中的高得多。这些结果表明，离子对或离子对网络的数量的增加有助于增加Pk-GK和Pk-RNase HII的稳定性，如对来自超嗜热古菌的其他酶所建议的。构建Pk-GK结构的三维模型并将其与大肠杆菌GK的晶体结构进行比较，支持了这一假设。我们现在正试图确定Pk-GK和Pk-RNase HII的晶体结构，并确定使这些酶比嗜温酶更稳定的氨基酸取代。

To understand the mechanism, by which the proteins from hyperthermophilic archaeon which grow at the temperature higher than 95ﾟC adapt unusually high temperatures, we have cloned the genes encoding glycerol kinase (GK) and ribonuclease HII (RNase HII) from Pyrococcus kodakaraensis KODI (Pk). these genes were overexpressed in E.coli and the recombinant enzymes were purified to homogeneity. Heat treatment was shown to be very effective to purify these enzymes, because most of the proteins from E.coli were precipitated upon heat treatment due to thermal denaturation and removed by centrifugation. Pk-GK and Pk-RNase HII are composed of 497 and 288 amino acid residues and exist in dimeric and monomeric forms, respectively. These enzymes show unusual enzyme characteristics, such as high stability against heat inactivation and broad metal ion specificity. Comparison of the amino acid compositions of these enzymes with those of the enzymes from mesophilic sources showed the contents of the charged amino acid residues in Pk-GK and Pk-RNase HII are much higher than those in E.coli GK and E.coli RNase HII,respectively. These results suggest that an increase in the number of the ion pairs or ion-pair networks contribute to increase the stabilities of Pk-GK and Pk-RNase HII,as suggested for other enzymes from hyperthermophilic archaea. Construction of a three-dimensional model for the Pk-GK structure and comparison of it with the crystal structure of E.coli GK supports this hypothesis. We are now trying to determine the crystal structures of Pk-GK and Pk-RNase HII,and to identify amino acid substitutions that makes these enzymes more stable than mesophilic ones.