An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability-activity trade-offs
An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability-activity trade-offs
复制标题
DOI：
10.1093/protein/gzm072
复制
发表时间：
2008-01-01
影响因子：
2.4
通讯作者：
Shamoo, Yousif
中科院分区：
文献类型：
--
作者：
Counago, Rafael;Wilson, Corey J.;Shamoo, Yousif
蛋白质的功能是活性和稳定性之间的平衡。然而，蛋白质进化的稳定性-活性权衡的相关性及其对生物体适应性的影响一直难以确定。在此之前，我们已经将生物体在温度升高下的生存与通过替换嗜热生物中必需基因腺苷酸激酶（adk）的等位基因对单个蛋白质序列的适应性变化联系起来。在体内对温度敏感的嗜热生物的持续进化表明，提高有机体适应性的第一步是中温酶（aksubq199r）中谷氨酰胺-199突变为精氨酸。在这里，我们表明，虽然取代Arg-199确实赋予了适度的稳定性增加(0.6千卡摩尔（-1）在20℃；δ T-m = 3.0℃)，在早期的实验进化研究中，酶的活性谱发生了很大的变化，这是其异常健壮性的原因。对AKsub Q199R的动力学研究表明，它在较低温度（20-45℃）下具有较强的酶活性损失（约50%），随后在高温下增加。AKsub Q199R的稳定性与活性之间的权衡关系与通过稳定含有Arg-199的多肽环（该多肽环是该酶的atp结合位点的一部分）而使其整体结构硬化有关。结构分析表明Arg-199促进了新的离子相互作用的形成。我们的研究结果表明，在自然选择过程中，稳定性和活动性的权衡很容易作为一种进化策略来提高生物体的适应性。
Protein function is a balance between activity and stability. However, the relevance of stability-activity trade-offs for protein evolution and their impact on organismal fitness have been difficult to determine. Previously, we have linked organismal survival at increasing temperatures to adaptive changes to a single protein sequence through allelic replacement of an essential gene, adenylate kinase (adk), in a thermophile. In vivo continuous evolution of the temperature-sensitive thermophile has shown that the first step toward increased organismal fitness is mutation of glutamine-199 to arginine in the mesophilic enzyme (AKsub Q199R). Here, we show that although substitution of Arg-199 did confer a modest increase in stability (0.6 kcal mol(-1)at 20 degrees C; Delta T-m = 3.0 degrees C), it is a large change in the activity profile of the enzyme that is responsible for its exceptional robustness during the earlier experimental evolution study. Kinetic studies of AKsub Q199R show that it has a strong loss of enzymatic activity (> 50%) at lower temperatures (20-45 degrees C) and a subsequent increase at elevated temperatures. The stability-activity trade-off observed for AKsub Q199R was linked to the rigidification of the overall structure through stabilization of a polypeptide loop containing Arg-199 that is part of the ATP-binding site of the enzyme. Structural analysis revealed the formation of new ionic interactions facilitated by Arg-199. Our results suggest that stability-activity trade-offs are employed readily as an evolutionary strategy during natural selection to increase organismal fitness.