安丝菌素聚酮合酶多功能DH机制解析与定向优化

安丝菌素因其极强的抗肿瘤活性，而在安莎类化合物中备受关注。像许多聚酮化合物一样，安丝菌素合成过程中会出现双键移位现象，即双键的位置从α-β位向β-γ位转移。研究发现PKS模块中脱水酶(dehydratase, DH)结构域可能具有脱水、双键迁移和甲基差向异构等多重功能，但其具体分子机制尚不明确。本项目拟从前期研究获得的安丝菌素DH2结构域晶体结构出发，构建DH结构域催化的计算模型，通过对底物识别、催化、释放全过程的机制解析，挖掘其中决定多功能的酶-底物关键作用位点，指导DH结构域的定向改造，以此拓展安丝菌素DH底物谱和提升催化效率，以期获取具有抗肿瘤活性的新结构化合物。本项目采用“干计算”与“湿实验”结合，集中体现合成生物学的“认知-设计-改造”理念，其结果将完善对PKS DH.结构域的功能认知，丰富聚酮化合物生物合成理论，为聚酮类化合物的合成生物学理性改造提供新的研究范式。

The ansamycins are important antibiotics, in which ansamitocins are regarded as outstanding anti-tumor drug precursors. The double bond shifts catalyzed by polyketide synthase (PKS) dehydratase (DH) domains occur during the biosynthesis of ansamycins, which means the position of the double bond shifted from α-β position to β-γ position. Some studies have reported that DH domains in PKS have multiple catalytic activities such as dehydration, double bond shift, and methyl epimerization. However, these underlying molecular mechanisms are still unclear. In this project, based on the available crystal structure of ansamycin DH2 domain, we intend to construct calculational models to study the catalytic mechanism of ansamycin DH domains (including dehydration and double bond shift) and to analyze the complete process of substrate recognition, enzymatic catalysis, and product release. Upon these analyses, the critical residues involved in the multiple catalytic activities of ansamycin DH domains will be identified. And subsequent directed engineering of DH domains may contribute to improving the catalytic efficiency of DH domains and broadening the substrate flexibility, which is of great significance for developing new antitumor derivatives. With the combination of "dry computation" and "wet-lab experiment", our results will provide mechanistic insight into the ansamycin DH domains and establish a new research paradigm for the rational design and optimization of key enzymes in polyketides biosynthesis.