基于柔性区域的计算设计改造玉米赤霉烯酮水解酶热稳定性

Computation-aided design of the flexible region of zearalenone hydrolase improves its thermal stability

摘要:

来源于粉红螺旋聚孢霉Clonostachys rosea的玉米赤霉烯酮水解酶 (ZHD101) 可以有效降解谷物农副产品和饲料中的霉菌毒素玉米赤霉烯酮 (Zearalenone，ZEN)，但是，该酶的热稳定性较低，限制了其在工业中的应用。由于水解ZEN的反应没有吸光值的变化，不适合高通量筛选。本文以ZHD101为模式酶，进行计算虚拟突变并结合实验验证。通过比对不同温度下的分子动力学模拟轨迹，选取32个柔性位点；再通过位置特异性评分和酶构象自由能计算，从32个柔性位点上的608个虚拟饱和突变体中筛选出12个突变体。经实验验证，其中3个突变体N156F、S194T和T259F的热熔融温度有一定程度的提升 (ΔTm>4 ℃)，且酶活性与野生型类似甚至更高 (相对酶活性为95.8%、131.6%和169.0%)。分子动力学模拟分析显示，导致3个突变体热稳定性提高的可能作用机理分别为NH-π作用力、盐桥重排和分子表面空穴填充。将3个突变体进行迭代组合突变，N156F/S194T表现出最高的热稳定性 (ΔTm=6.7 ℃)。这项工作表明基于柔性区域的虚拟饱和突变在酶稳定性改造上的可行性，探索计算虚拟改造结合实验验证的酶改造策略。

Abstract:

The zearalenone hydrolase (ZHD101) derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone (ZEN) present in grain by-products and feed. However, the low thermal stability of ZHD101 hampers its applications. High throughput screening of variants using spectrophotometer is challenging because the reaction of hydrolyzing ZEN does not change absorbance. In this study, we used ZHD101 as a model enzyme to perform computation-aided design followed by experimental verification. By comparing the molecular dynamics simulation trajectories of ZHD101 at different temperatures, 32 flexible sites were selected. 608 saturated mutations were introduced into the 32 flexible sites virtually, from which 12 virtual mutants were screened according to the position specific score and enzyme conformation free energy calculation. Three of the mutants N156F, S194T and T259F showed an increase in thermal melting temperature (ΔTm>4 °C), and their enzyme activities were similar to or even higher than that of the wild type (relative enzyme activity 95.8%, 131.6% and 169.0%, respectively). Molecular dynamics simulation analysis showed that the possible mechanisms leading to the improved thermal stability were NH-π force, salt bridge rearrangement, and hole filling on the molecular surface. The three mutants were combined iteratively, and the combination of N156F/S194T showed the highest thermal stability (ΔTm=6.7 °C). This work demonstrated the feasibility of engineering the flexible region to improve enzyme performance by combining virtual computational mutations with experimental verification.