Haruki Mitsuru, Saito Yoshitaka, Ota Motonori, Nishikawa Ken, Kanaya Shigenori
Department of Materials Chemistry and Engineering, College of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan.
J Biotechnol. 2006 Jul 25;124(3):512-22. doi: 10.1016/j.jbiotec.2006.01.022. Epub 2006 Mar 20.
The change in the structural stability of Escherichia coli ribonuclease HI (RNase HI) due to single amino acid substitutions has been estimated computationally by the stability profile of mutant protein (SPMP) [Ota, M., Kanaya, S. Nishikawa, K., 1995. Desk-top analysis of the structural stability of various point mutations introduced into ribonuclease H. J. Mol. Biol. 248, 733-738]. As well, an effective strategy using random mutagenesis and genetic selection has been developed to obtain E. coli RNase HI mutants with enhanced thermostability [Haruki, M., Noguchi, E., Akasako, A., Oobatake, M., Itaya, M., Kanaya, S., 1994. A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions. J. Biol. Chem. 269, 26904-26911]. In this study, both methods were combined: random mutations were individually introduced to Lys99-Val101 on the N-terminus of the alpha-helix IV and the preceding beta-turn, where substitutions of other amino acid residues were expected to significantly increase the stability from SPMP, and then followed by genetic selection. Val101 to Ala, Gln, and Arg mutations were selected by genetic selection. The Val101-->Ala mutation increased the thermal stability of E. coli RNase HI by 2.0 degrees C in Tm at pH 5.5, whereas the Val101-->Gln and Val101-->Arg mutations decreased the thermostability. Separately, the Lys99-->Pro and Asn100-->Gly mutations were also introduced directly. The Lys99-->Pro mutation increased the thermostability of E. coli RNase HI by 1.8 degrees C in Tm at pH 5.5, whereas the Asn100-->Gly mutation decreased the thermostability by 17 degrees C. In addition, the Lys99-->Pro mutation altered the dependence of the enzymatic activity on divalent metal ions.
通过突变蛋白稳定性图谱（SPMP）[太田真、金谷幸、西川和夫，1995年。核糖核酸酶H中各种点突变的结构稳定性的桌面分析。《分子生物学杂志》248卷，733 - 738页]，已通过计算估计了由于单个氨基酸替换导致的大肠杆菌核糖核酸酶HI（RNase HI）结构稳定性的变化。此外，还开发了一种利用随机诱变和基因筛选的有效策略，以获得热稳定性增强的大肠杆菌RNase HI突变体[春木真、野口惠、赤坂晃、大畑正、板谷正、金谷幸，1994年。一种稳定大肠杆菌核糖核酸酶HI的新策略，涉及筛选羧基末端缺失的基因内抑制子。《生物化学杂志》269卷，26904 - 26911页]。在本研究中，将两种方法结合起来：在α - 螺旋IV的N端和前面的β - 转角处的Lys99 - Val101上分别引入随机突变，预计其他氨基酸残基的替换会根据SPMP显著提高稳定性，然后进行基因筛选。通过基因筛选选择了Val101突变为Ala、Gln和Arg的突变体。在pH 5.5时，Val101→Ala突变使大肠杆菌RNase HI的热稳定性在熔点温度（Tm）下提高了2.0℃，而Val101→Gln和Val101→Arg突变则降低了热稳定性。另外，还直接引入了Lys99→Pro和Asn100→Gly突变。在pH 5.5时，Lys99→Pro突变使大肠杆菌RNase HI的热稳定性在Tm下提高了1.8℃，而Asn100→Gly突变使热稳定性降低了17℃。此外，Lys99→Pro突变改变了酶活性对二价金属离子的依赖性。
