几个新颖[4+2]环加成酶的结构及其催化机制研究

The Diels-Alder [4+2] cycloaddition is one of the most classic and widely used chemical reactions in synthetic chemistry. However, the key question of whether a Diels-Alderase occurs naturally is currently still under debate between chemist and biologist, and remain unsolved for decades. Recently, during the investigation of the biogenesis of spirotetronate/spirotetramate natural products such as pyrroindomycins, people have identified four novel enzymes, PyrE3, Pyrl4, PloI4 and QmnH, which are demonstrated to catalyze four different [4+2] cycloaddition reactions. However, the structures and the detailed enzymatic mechanism of these enzymes are still largely unknown. In this research proposal, by using multidisciplinary approaches including structure biology, biochemistry and computer simulations methods, we want to systemically characterize the structures and the detailed molecular mechanism governing the enzymatic functions of these enzymes, thereby to elucidate how these enzymes catalyze different [4+2] cycloaddition reactions, and to address the long-standing question whether there are natural enzymes catalyzing Diels-Alders reactions. Meanwhile, based on their solved structures, we also want to modify and engineer these enzymes to improve their enzymatic properties for the future synthetic biology study and for the potential application research.

狄尔斯-阿尔德(Diels-Alder)反应是合成化学领域中最经典和最被广泛应用的反应类型之一。然而，在自然界中是否存在能够催化狄尔斯-阿尔德反应的蛋白酶，长期以来一直是化学家和生物学家争论且悬而未决的焦点科学问题。近期，人们在研究螺环乙酰乙酸内酯/内酰胺类抗生素的生源合成的过程中发现了四个可以催化不同[4+2]环加成反应的新颖蛋白酶，PyrE3、Pyrl4、PloI4和QmnH。但是，目前这些新颖蛋白酶的结构及其相关的催化机制绝大部分仍然未知。本申请课题计划综合采用结构生物学、生物化学、计算模拟等方法来系统地研究上述新颖蛋白酶的结构和催化不同[4+2]环加成反应的分子机制，并验证它们催化的[4+2]环加成反应是否为狄尔斯-阿尔德反应，从而来证明自然界狄尔斯-阿尔德酶的存在。同时，我们也将在结构的基础上改造上述蛋白酶，使它们具有更好的催化性能，并用于后续相关的合成生物学和潜在的应用研究。

Diels-Alder反应是合成化学领域中最经典和最被广泛应用的反应类型之一。然而，自然界中是否存在天然的Diels-Alder酶及其具体的催化机制一直是领域内悬而未决的关键科学问题。PyrE3、Pyrl4、PloI4和QmnH是螺环乙酰乙酸内酯/内酰胺类抗生素生源合成过程中催化不同[4+2]环加成反应的四个新颖蛋白酶，但其三维结构和催化机制未知，同时它们催化的[4+2]环加成反应是否为Diels-Alder反应也未明。本项目综合采用结构生物学、化学生物学、生物化学等方法对这几个新颖蛋白酶的三维结构和催化机制进行了系统的研究。项目执行期间，首次解析了新颖[4+2]环加成酶PyrE3、Pyrl4、PloI4和相关的TsrL的三维结构，详细阐明了其中PyrE3、Pyrl4、PloI4催化不同[4+2]环加成反应的分子机制，并证实PyrE3、Pyrl4、PloI4催化的[4+2]环加成反应是Diels-Alder反应。此外，基于Pyrl4和PloI4的结构信息，我们成功实现了PloI4的功能改造，获得了催化性能改良的PloI4突变体，并成功用于螺环乙酰乙酸内酯/内酰胺类抗生素的新分子结构的创制。总之，本项目的研究为后续相关的化学生物学和合成生物学研究提供了重要的科学基础。