基于计算库设计的稳健的助溶剂相容卤代醇脱卤酶

Hesam Arabnejad, Marco Dal Lago, P. Jekel, Robert J. Floor, A. Thunnissen, A. C. Terwisscha van Scheltinga, Hein J. Wijma, D. Janssen
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引用次数: 34

摘要

为了提高卤代醇脱卤酶在有机共溶剂存在下作为反应催化剂的适用性,我们探索了一种计算库设计策略(计算库快速酶稳定框架),包括发现和计算机评估稳定突变。能量计算、二硫化物键预测和分子动力学模拟确定了218个点突变和35个二硫化物键具有预测的稳定作用。实验证实了29个稳定点突变,其中大部分位于两个不同的区域,而引入二硫键则无效。将最佳突变组合在一起,得到了12倍突变体(HheC-H12),表观熔融温度提高28°C,对共溶剂的抗性显著提高。该突变体表现出较高的最适催化温度,同时保持了低温下的活性。突变体H12被用作模板,用于引入增强对映体选择性或活性的突变。晶体结构表明,H12突变体的结构变化与计算预测基本一致,稳定性的增强主要是由于突变重新分配了表面电荷和改善了亚基之间的相互作用,后者包括更好的水分子在亚基界面的相互作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A robust cosolvent-compatible halohydrin dehalogenase by computational library design
To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.
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