真菌聚酮合酶的结构功能分析

The fungi polyketide synthases (PKSs) are the large multifunctional proteins that produce polyketide secondary metabolites with complicated structures and diverse bioactivities. During the biosynthesis of polyketides, the acyl carrier protein (ACP) domains cooperate with the catalysis domains to determine the efficiency and stereospecificity of the catalysis process. However, the poor understanding of molecular mechanism in individual domain catalysis process and protein-protein interaction between ACP and the catalysis domains had posed a significant challenge to engineering efforts. pksA and pksCT, which responsible for aflatoxin and citrinin biosynthesis in fungi repectively, were chosen for our previous study. We had expressed and purified all the individual domains in these two PKSs, and obtained the crystals of three domains (starter and extender acyltransferase (AT) domains from pksCT, and extender AT domain from pksA). In this proposal, we will focus on 1) solving the crystal structures of individual domains to identify key residues involved in catalysis process; 2) quantifying the interactions between ACP and partner domains to reveal the roles of ACP in catalysis process by using the fluorescence polarization method; 3) preparing stable ACP-partner domain complexes by mechanism based cross-linking, and solving the crystal structures of protein complexes to identify key residues involved in ACP–partner domain interaction.

真菌聚酮合酶（PKS）是由多个催化结构域组成的多功能蛋白，催化合成一大类结构复杂、活性多样的聚酮产物，合酶中酰基载体蛋白（ACP）和各个催化结构域互作共同决定催化反应速率和特异性，但目前合酶中单个催化结构域的结构功能、结构域间互作的报道都较少，限制了其设计和改造。前期工作中我们以参与合成黄曲霉毒素的pksA和参与桔霉素合成的pksCT两个真菌PKS为研究对象，已表达和纯化了所有单结构域，并且获得pksA的延伸酰基转移酶（AT）结构域以及pksCT起始和延伸AT结构域的晶体。本研究中我们将1）解析单个结构域晶体结构，鉴定结构域中影响催化反应的关键位点；2）利用荧光偏振等技术快速、高通量测定ACP和各结构域的相互作用，分析ACP对催化反应的影响；3）利用基于催化机制的共价交联方法获得ACP 和催化结构域的稳定复合物，解析其晶体结构，鉴定参与结构域间相互作用的位点。

聚酮合酶（PKS）是由多个催化结构域组成的多功能蛋白，催化合成一大类结构复杂、活性多样的聚酮产物，合酶中酰基载体蛋白（ACP）和各个催化结构域互作共同决定催化反应速率和特异性，但目前合酶中结构域间互作的报道较少，限制了其设计和改造。本研究将综合利用结构生物学、生物化学、分子生物学以及生物信息学等手段对ACP和各个催化结构域相互作用共同决定催化速率和特异性的分子机理进行系统探索。研究的主要结果如下：.基于系统发生树分析发现，来源于真菌迭代型PKS的酰基转移酶（AT）和同样以丙二酰CoA为底物的细菌模块型PKS的AT可以明显划分为4组，进一步晶体结构和催化中心保守模序的比较发现了4组AT的保守型和差异性。.利用基于催化机制的共价交联方法来获得了ACP 和硫酯酶催化结构域的稳定复合物，本研究中发现单个结构域之间难以实现显著交联，而采用融合蛋白的方法可以大幅度提高两者的交联效率。.利用荧光偏振技术来高通量测定ACP和催化结构域之间的相互作用，已建立了荧光分子稳定标记聚酮合酶ACP结构域的方法。