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首页> 外文期刊>Journal of Molecular Biology >Evolution of stability in a cold-active enzyme elicits specificity relaxation and highlights substrate-related effects on temperature adaptation.
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Evolution of stability in a cold-active enzyme elicits specificity relaxation and highlights substrate-related effects on temperature adaptation.

机译：在冷活性酶中稳定性的演变引起特异性松弛，并突出了底物对温度适应的影响。

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摘要

Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 degrees C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity.
机译：通过遵循温度依赖性，构象稳定性和通过定向进化修饰的冷活性脂肪酶中底物特异性的共同进化，研究了蛋白质热适应的分子方面。我们发现动力学稳定性的演变伴随着底物特异性的松弛。而且，事实证明温度依赖性和选择性是相互依赖性的。尽管野生型蛋白在10-50摄氏度的温度范围内对短链甘油三酸酯（C4）严格具有特异性，并在寒冷下表现出最高的活性，但其稳定的变体能够接受C8和C12分子，其选择性为温度依赖。当结构受到温度或化学变性剂的挑战时，我们无法检测到突变体整体结构坚固性的任何改善。但是，有强有力的证据表明，通过两种独立的方法可以在活动站点区域实现局部稳定效果。差示扫描荧光法显示，疏水性斑块的暴露（如活性位点一样）先于变性，分子动力学模拟证实，通过限制盖的活动性即可获得稳定性，盖的活动性是调节进入酶活性位点的灵活结构并影响其稳定性。盖运动的这种减少被认为伴随着其他蛋白质区域的迁移率的增加，因此解释了观察到的底物特异性的拓宽。

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