Kobayashi Miyuki, Takada Yasuhiro
Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku, Sapporo, 060-0810, Japan.
Extremophiles. 2014 Jul;18(4):755-62. doi: 10.1007/s00792-014-0656-7. Epub 2014 Jun 10.
In the two cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH and CpIDH, respectively), the combined substitutions of amino acid residues between the Leu693, Leu724 and Phe735 residues of CmIDH and the corresponding Phe693, Gln724 and Leu735 residues of CpIDH were introduced by site-directed mutagenesis. A double mutant of CmIDH substituted its Leu724 and Phe735 residues by the corresponding ones of CpIDH, CmL724Q/F735L, and the triple mutant of CpIDH, CpF693L/Q724L/L735F, showed the most decrease and increase of activity, respectively, of each wild-type and its all mutated enzymes. In the case of CmIDH, the substitutions of these three amino acid residues resulted in the decrease of catalytic activity and thermostability for activity, but the combined substitutions of amino acid residues did not necessarily exert additive effects on these properties. On the other hand, similar substitutions in CpIDH had quite opposite effects to CmIDH, and the effects of the combined substitutions were additive. All multiple mutants of CmIDH and CpIDH showed lower and higher catalytic efficiency (k(cat)/K(m)) values than the respective wild-type enzymes. Single and multiple mutations of the substituted amino acid residues in the CmIDH and CpIDH led to the increase and decrease of sensitivity to tryptic digestion, indicating that the stability of protein structure was decreased and increased by the mutations, respectively.
在来自嗜冷细菌马里氏科尔韦氏菌和嗜冷红科尔韦氏菌的两种冷适应性单体异柠檬酸脱氢酶（分别为CmIDH和CpIDH）中，通过定点诱变引入了CmIDH的Leu693、Leu724和Phe735残基与CpIDH相应的Phe693、Gln724和Leu735残基之间的氨基酸残基组合替换。CmIDH的一个双突变体将其Leu724和Phe735残基替换为CpIDH的相应残基，即CmL724Q/F735L，而CpIDH的三突变体CpF693L/Q724L/L735F分别在每种野生型及其所有突变酶中表现出最大程度的活性降低和增加。就CmIDH而言，这三个氨基酸残基的替换导致催化活性和活性热稳定性降低，但氨基酸残基的组合替换不一定对这些特性产生累加效应。另一方面，CpIDH中的类似替换对CmIDH产生了完全相反的影响，并且组合替换的影响是累加的。CmIDH和CpIDH的所有多突变体均显示出比各自野生型酶更低和更高的催化效率（k(cat)/K(m)）值。CmIDH和CpIDH中被替换氨基酸残基的单突变和多突变分别导致对胰蛋白酶消化的敏感性增加和降低，这表明突变分别降低和增加了蛋白质结构的稳定性。
