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首页> 外文期刊>The FEBS journal >Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation
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Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation

机译：增强了Ochrobactrum sp。的甲基对硫磷水解酶的热稳定性。 M231通过合理改造甘氨酸来脯氨酸突变

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Protein thermostability can be increased by some glycine to proline mutations in a target protein. However, not all glycine to proline mutations can improve protein thermostability, and this method is suitable only at carefully selected mutation sites that can accommodate structural stabilization. In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved by the experiments. The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 (Ochr-MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure. A profile of the root mean square fluctuations of Ochr-MPH was calculated at the nanosecond timescale, and an eight-amino acid loop region (residues 186-193) was identified as having high conformational fluctuation. The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity. The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P/G198P) were modeled and analyzed using molecular dynamics simulations. The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr-MPH. The thermostability and kinetic behavior of the wild-type and three mutant enzymes were measured. The results were consistent with the computational predictions, and the mutant G194P was found to have higher thermostability than the wild-type enzyme.
机译：某些甘氨酸可增加蛋白质热稳定性，使目标蛋白质中的脯氨酸突变。但是，并非所有甘氨酸到脯氨酸的突变都可以提高蛋白质的热稳定性，并且该方法仅适用于精心选择的可以适应结构稳定的突变位点。在这项研究中，同源性建模和分子动力学模拟被用来选择合适的甘氨酸脯氨酸突变，以提高蛋白质的热稳定性，并通过实验证明了所选突变的效果。 Ochrobactrum sp。的甲基对硫磷水解酶（MPH）的结构。通过同源性建模构建M231（Ochr-MPH），并在建模的结构上进行分子动力学模拟。在纳秒时间尺度上计算了Ochr-MPH的均方根波动的轮廓，并确定了一个八个氨基酸的环状区域（残基186-193）具有高构象波动。选择距离该区域最近的两个甘氨酸作为可能影响附近蛋白质柔韧性的突变目标。使用分子动力学模拟对两个单突变体（G194P和G198P）和一个双突变体（G194P / G198P）的结构和构象波动进行建模和分析。结果表明，突变体G194P在环区的构象波动减少，可能增加了Ochr-MPH的热稳定性。测量了野生型和三种突变酶的热稳定性和动力学行为。结果与计算预测一致，并且发现突变体G194P具有比野生型酶更高的热稳定性。

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