Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
Institute of Animal Science and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou 571100, P. R. China.
Sci Rep. 2016 Dec 8;6:38413. doi: 10.1038/srep38413.
Understanding the dynamics of the key pectinase, polygalacturonase, and improving its thermotolerance and catalytic efficiency are of importance for the cost-competitive bioconversion of pectic materials. By combining structure analysis and molecular dynamics (MD) simulations, eight mutagenesis sites having the potential to form cation-π interactions were identified in the widely used fungal endo-polygalacturonase PG63. In comparison to the wild-type, three single mutants H58Y, T71Y and T304Y showed improved thermostability (the apparent Ts increased by 0.6-3.9 °C) and catalytic efficiency (by up to 32-fold). Chromatogram analysis of the hydrolysis products indicated that a larger amount of shorter sugars were released from the polygalacturonic acid by these three mutants than by the wild-type. MD analysis of the enzyme-substrate complexes illustrated that the mutants with introduced cation-π interaction have modified conformations of catalytic crevice, which provide an enviable environment for the catalytic process. Moreover, the lower plasticity of T3 loop 2 at the edge of the subsite tunnel appears to recruit the reducing ends of oligogalacturonide into the active site tunnel and initiates new hydrolysis reactions. This study demonstrates the importance of cation-π interaction in protein conformation and provides a realistic strategy to enhance the thermotolerance and catalytic performance of endo-polygalacturonases.
理解关键果胶酶、多聚半乳糖醛酸酶的动态，并提高其耐热性和催化效率，对于具有成本竞争力的果胶材料的生物转化至关重要。通过结构分析和分子动力学（MD）模拟相结合，在广泛使用的真菌内切多聚半乳糖醛酸酶 PG63 中鉴定出了 8 个具有形成阳离子-π 相互作用潜力的突变位点。与野生型相比，三个单突变体 H58Y、T71Y 和 T304Y 表现出更好的热稳定性（表观 Ts 提高了 0.6-3.9°C）和催化效率（提高了 32 倍）。水解产物的色谱分析表明，这三个突变体从多聚半乳糖醛酸中释放出的短糖量比野生型多。对酶-底物复合物的 MD 分析表明，引入阳离子-π 相互作用的突变体改变了催化裂缝的构象，为催化过程提供了一个令人羡慕的环境。此外，位于亚位点隧道边缘的 T3 环 2 的较低可塑性似乎将寡半乳糖醛酸的还原端募集到活性位点隧道中，并引发新的水解反应。这项研究表明了阳离子-π 相互作用在蛋白质构象中的重要性，并提供了一种增强内切多聚半乳糖醛酸酶耐热性和催化性能的现实策略。
