对环氧化合物水解酶Lsd19的突变研究和催化定向改造

The crystal structure of Lsd19, an epoxide hydrolase enzyme, in complex with the natural product Lasalocid A and substrate analog revealed the mechanism of anti-Baldwin ring closure reaction responsible for polyether biosynthesis. This study is a breakthrough for natural product biosynthesis. In our investigation we will conduct mutagenesis studies to further improve our understanding of the catalytic mechanism. Furthermore, we will engineer new Lsd19A and Lsd19B domains which catalyze the first exo ring and second endo ring formation respectively. The function of Lsd19A will be changed to catalyze the second exo ring formation and the function of Lsd19B will be changed to catalyze the first endo ring formation. Using different combinations of these domains, we will be able to biosynthesize new Lasalocid A analogs containing 5-exo and 5-exo, 6-endo and 6-exo, 6-endo and 7-endo rings. This approach will enable us synthesize any exo or endo ring for polyether, thereby significantly increasing the pool of natural product-like compounds for drug discovery. This is a novel approach for natural product drug development.

环氧化合物水解酶Lsd19与天然产物Lasalocid A以及底物类似物的晶体结构阐释了聚合醚水解酶生成反Baldwin规则endo环的催化机理，是天然化合物生物合成机制的一大突破。本项目延续这一研究，通过氨基酸突变测试参与催化的氨基酸的功能，进一步印证反应机制。此外，本项目根据Lsd19结构改造其两个结构域Lsd19A与Lsd19B(Lsd19A催化生成第一个5元exo环，Lsd19B催化生成第二个6元endo环)，使Lsd19A合成第二个exo环，而Lsd19B合成第一个endo环。经过这些结构域的排列组合，本项目拟得到Lasalocid A的类似物包括5-exo-5-exo环，6-endo-6-exo环，6-endo-7-endo环。这项技术可以促进任意合成聚合醚的exo环或endo环，从而扩大了类天然化合物药物的数据库，是天然化合物药物研发的首创。

Lsd18与Lsd19是合成天然产物Lasalocid A的关键酶。Lsd18氧化双键形成环氧化合物，Lsd19进一步水解该环氧化物形成环状醚。本项目有机合成了Lsd18的天然底物的类似物，Lsd18的天然产物的类似物（即Lsd19的底物），解析了Lsd18与底物，产物的复合体的晶体结构，阐明了Lsd18的催化机制，并对Lsd18进行改造，提高了其稳定性。本项目对Lsd19也进行了改造，设计，表达，纯化了Lsd19A，Lsd19B，Lsd19A G104M和Lsd19B M235G突变体，进行了初步的酶活测试。该研究对于将酶催化应用于有机合成具有重要意义。