Entropic contribution to enhanced thermal stability in the thermostable P450 CYP119.
Entropic contribution to enhanced thermal stability in the thermostable P450 CYP119.
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熵对增强热稳定 P450 CYP119 热稳定性的贡献
DOI：
10.1073/pnas.1807473115
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发表时间：
2018-10-23
影响因子：
11.1
通讯作者：
Jain N
中科院分区：
文献类型：
--
作者：
Liu Z;Lemmonds S;Huang J;Tyagi M;Hong L;Jain N
了解导致嗜热细胞色素 P450 酶稳定性增强的热力学因素对于将其开发为高温单氧化反应的有效催化剂具有重要意义。这些因素通常是通过与嗜温 P450 的结构比较来推断的，并调用刚性化的焓相互作用作为热稳定性的主要热力学决定因素。然而，以嗜热 P450 CYP119 为例，目前的工作对 P450 热稳定性的这种由焓驱动的概念提出了质疑，并提供了强有力的实验证据，表明它们的热稳定性实际上可能是熵驱动的，因为相对于中温 P450 折叠状态的灵活性增加，并且这种灵活性在功能活性和热稳定性之间有效分配。这代表了理解嗜热 P450 热稳定性基础的重大范式转变。与嗜温蛋白质相比，嗜热蛋白质的热稳定性增强通常归因于蛋白质残基之间的强烈相互作用所产生的焓效应。直观上，这些强烈的残基间相互作用将使生物分子刚性化。然而，目前利用中子散射和溶液核磁共振波谱测量的工作证明了一个相反的例子，即嗜热细胞色素 P450（CYP119）比其嗜温对应物 CYP101A1 更加灵活，这一点仅从两种蛋白质的结构比较中并不明显。解释这一明显矛盾的机制是，CYP119折叠状态的较高灵活性增加了其构象熵，从而减少了变性过程中的熵增益，这将增加解折叠所需的自由能，从而稳定蛋白质。这种情况得到了关于展开自由能的温度依赖性的热力学数据的支持，该数据显示了熵对 CYP119 的热稳定性的显着贡献，并为增强稳定性提供了一个额外的维度，之前仅归因于芳香堆积相互作用和盐桥网络的存在。我们的实验数据还支持这样的观点：高度嗜热的 P450（例如 CYP119）可能使用一种机制，在配体结合和热稳定性之间分配与嗜温 P450 不同的灵活性。
Understanding the thermodynamic factors responsible for enhanced stability of thermophilic cytochrome P450 enzymes is significant in their development as efficient catalysts for high-temperature monooxygenation reactions. These factors are usually inferred from structural comparison with mesophilic P450s and invoke rigidifying enthalpic interactions as primary thermodynamic determinants for thermostability. Using the thermophilic P450 CYP119 as an example, the present work, however, questions this enthalpy-driven notion of P450 thermostability and provides strong experimental evidence that their thermostability may actually be entropy-driven due to increased flexibility in the folded state relative to mesophilic P450s and that this flexibility is partitioned effectively between functional activity and thermal stability. This represents a major paradigm shift in understanding the basis of thermostability in thermophilic P450s. The enhanced thermostability of thermophilic proteins with respect to their mesophilic counterparts is often attributed to the enthalpy effect, arising from strong interactions between protein residues. Intuitively, these strong interresidue interactions will rigidify the biomolecules. However, the present work utilizing neutron scattering and solution NMR spectroscopy measurements demonstrates a contrary example that the thermophilic cytochrome P450, CYP119, is much more flexible than its mesophilic counterpart, CYP101A1, something which is not apparent just from structural comparison of the two proteins. A mechanism to explain this apparent contradiction is that higher flexibility in the folded state of CYP119 increases its conformational entropy and thereby reduces the entropy gain during denaturation, which will increase the free energy needed for unfolding and thus stabilize the protein. This scenario is supported by thermodynamic data on the temperature dependence of unfolding free energy, which shows a significant entropic contribution to the thermostability of CYP119 and lends an added dimension to enhanced stability, previously attributed only to presence of aromatic stacking interactions and salt bridge networks. Our experimental data also support the notion that highly thermophilic P450s such as CYP119 may use a mechanism that partitions flexibility differently from mesophilic P450s between ligand binding and thermal stability.
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影响因子：
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通讯作者：
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作者：
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