Álvarez-Cao María-Efigenia, González Roberto, Pernas María A, Rúa María Luisa
Department of Food and Analytical Chemistry, Sciences Faculty of Ourense, University of Vigo, As Lagoas s/n, 32004 Ourense, Spain.
Microorganisms. 2018 Oct 19;6(4):108. doi: 10.3390/microorganisms6040108.
Thermophilic proteins have evolved different strategies to maintain structure and function at high temperatures; they have large, hydrophobic cores, and feature increased electrostatic interactions, with disulfide bonds, salt-bridging, and surface charges. Oligomerization is also recognized as a mechanism for protein stabilization to confer a thermophilic adaptation. Mesophilic proteins are less thermostable than their thermophilic homologs, but oligomerization plays an important role in biological processes on a wide variety of mesophilic enzymes, including thermostabilization. The mesophilic yeast contains a complex family of highly related lipase isoenzymes. Lip3 has been purified and characterized in two oligomeric states, monomer (mLip3) and dimer (dLip3), and crystallized in a dimeric conformation, providing a perfect model for studying the effects of homodimerization on mesophilic enzymes. We studied kinetics and stability at different pHs and temperatures, using the response surface methodology to compare both forms. At the kinetic level, homodimerization expanded Lip3 specificity (serving as a better catalyst on soluble substrates). Indeed, dimerization increased its thermostability by more than 15 °C (maximum temperature for dLip3 was out of the experimental range; >50 °C), and increased the pH stability by nearly one pH unit, demonstrating that oligomerization is a viable strategy for the stabilization of mesophilic enzymes.
嗜热蛋白已经进化出不同的策略来在高温下维持结构和功能；它们具有大的疏水核心，并具有增强的静电相互作用，包括二硫键、盐桥和表面电荷。寡聚化也被认为是一种蛋白质稳定化机制，可赋予嗜热适应性。中温蛋白的热稳定性低于其嗜热同源物，但寡聚化在多种中温酶的生物过程中起着重要作用，包括热稳定化。中温酵母含有一个高度相关的脂肪酶同工酶复杂家族。Lip3已被纯化并表征为两种寡聚状态，单体（mLip3）和二聚体（dLip3），并以二聚体构象结晶，为研究同源二聚化对中温酶的影响提供了一个完美的模型。我们使用响应面方法在不同pH值和温度下研究了动力学和稳定性，以比较两种形式。在动力学水平上，同源二聚化扩展了Lip3的特异性（对可溶性底物是更好的催化剂）。实际上，二聚化使其热稳定性提高了超过15℃（dLip3的最高温度超出实验范围；>50℃），并使pH稳定性提高了近一个pH单位，表明寡聚化是稳定中温酶的一种可行策略。
