Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation

文献类型： 外文期刊

第一作者： Tian, Jian

作者： Tian, Jian;Wang, Ping;Gao, Shan;Chu, Xiaoyu;Wu, Ningfeng;Fan, Yunliu

作者机构：

关键词： proline: 609-36-9;mutation;glycine: 56-40-6;mutation;methyl parathion hydrolase: MPH;406722-38-1;G194P;G198P;enzyme activity;molecular dynamics;kinetic behavior

期刊名称：FEBS JOURNAL （ 影响因子：5.542； 五年影响因子：5.5 ）

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收录情况： SCI

摘要： 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.

分类号： Q5

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