Thermostability Mechanism for the Hyperthermophilicity of Extremophile Cellulase TmCel12A: Implied from Molecular Dynamics Simulation.
Thermostability Mechanism for the Hyperthermophilicity of Extremophile Cellulase TmCel12A: Implied from Molecular Dynamics Simulation.
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DOI：
10.1021/acs.jpcb.6b03782
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发表时间：
2016-07
期刊：
影响因子：
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通讯作者：
P. Lian;C. Yuan;Qin Xu;W. Fu
中科院分区：
文献类型：
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作者：
P. Lian;C. Yuan;Qin Xu;W. Fu
纤维素酶的热稳定性对纤维素乙醇的生产具有重要意义。超嗜热菌海栖热袍菌（Thermotoga maritima）纤维素酶12 A（TmCel 12 A）是研究纤维素酶热稳定性的理想菌株。野生型酶的最适温度为85 °C。最近，已经观察到表面环突变Y 61 G不仅加快了水解速率，而且延长了酶在高温下的半衰期。然而，Y 61 G如何增强TmCel 12 A的热稳定性的机制尚未被揭示。本文采用分子动力学模拟和动态关联网络分析相结合的方法对TmCel 12 A的热稳定性机理进行了探讨。由Y 61、W176、V62和L144在结合口袋中构建的疏水簇被发现在调节TmCel 12 A的热稳定性以及催化能力中起关键作用。它在高温下稳定脱辅基酶;然而，它阻碍底物结合。Y 61 G突变干扰疏水簇，因为对应的氨基酸W176与R60形成阳离子-π相互作用，而不是WT中与Y 61的π-π相互作用。此外，Y 61 G突变通过改变酶铰链部分的氨基酸群落使酶更刚性和更延伸。我们的模拟结果也证实了早期从晶体学观察中提出的Y 61 G可能加速产物释放的假设。这些发现为理论和实验研究人员提供了一个新的方向，以提高其他纤维素酶的热稳定性，可以在生物燃料工业中的潜在应用。
Thermostability is of considerable importance for the application of cellulase in cellulosic ethanol production. The cellulase 12A from the hyperthermophile Thermotoga maritima (TmCel12A) is an ideal candidate to study thermostability of cellulases. Optimal temperature of the wild-type enzyme is 85 °C. Recently, it has been observed that surface loop mutation Y61G not only accelerates the hydrolysis rate but also extends the half-life of the enzyme at high temperature. However, the mechanism of how Y61G enhances thermostability of TmCel12A has not been revealed. Here, molecular dynamics simulation together with dynamic correlation network analysis was used to explore thermostability mechanism of TmCel12A. A hydrophobic cluster constructed by Y61, W176, V62, and L144 in the binding pocket was found to play a pivotal role in modulating thermostability as well as catalytic capability of TmCel12A. It stabilizes the apoenzyme at high temperature; however, it impedes the substrate binding. Y61G mutation disturbs the hydrophobic cluster as the counterpart amino acid W176 forms a cation-π interaction with R60 instead of the π-π interaction with Y61 in WT. Moreover, Y61G mutation makes the enzyme more rigid and more extended via altering the amino acid communities at the hinge part of the enzyme. An earlier hypothesis proposed from crystallographic observation that Y61G may accelerate the products releasing has been also confirmed by our simulations. These findings may provide a new direction for both theoretical and experimental scientists to improve the thermostability of other cellulases that can be potentially applied in biofuel industry.