模块化聚酮合酶(PKS)是功能强大的生物合成工厂，催化合成结构复杂、活性多样的聚酮次级代谢产物。生物合成研究表明，酮基还原酶(KR)和酰基载体蛋白(ACP)结构域相互协作共同决定新生聚酮链α-甲基和β-羟基手性中心的立体构型。前期工作中我们解析了不同类型KR的晶体结构，并通过功能分析鉴定其催化中心影响酮基还原产物立体构型的关键位点,但ACP如何通过蛋白相互作用影响KR的催化效率和立体特异性仍不清楚。本研究中我们将1)通过基于催化机制的共价交联获得KR和ACP的稳定复合物；2)解析复合物的晶体结构，鉴定参与结构域间蛋白相互作用的位点，揭示ACP结合引起的KR催化中心构象变化，比较不同类型KR和ACP之间的结合位点的差异；3)定点突变上述结构分析确定的关键位点，体外功能分析验证突变对酮基还原反应的影响；4)对PKS的KR催化中心及KR和ACP相互作用的位点进行突变，实现产物手性中心的构型翻转。

Modular polyketide synthases (PKSs) are versatile biosynthetic assembly lines responsible for the synthesis of polyketide secondary metabolites with complicated structures and diverse bioactivities. During the biosynthesis of polyketides, the ketoreductase (KR) and acyl carrier protein (ACP) domains cooperate with each other to set the stereochemistry of α-methyl and β-hydroxyl groups of nascent polyketide chains. In previous research, we have solved crystal structures of different type of KRs and identified key active site residues that affect the stereochemistry of reduced products, whereas how ACPs affect the efficiency and stereospecificity of KRs by protein-protein interactions is still unclear. In this proposal, we will focus on 1) preparing stable KR-ACP complexes by mechanism based cross-linking; 2) solving the crystal structures of protein complexes to identify key residues involved in KR-ACP interactions, to reveal the structural alterations of active sites of KRs upon binding of ACPs, and to compare the difference of ACP binding sites of various KRs; 3) verifying the effects of these residues on ketoreduction reactions by site-directed mutagenesis and in vitro functional assays; and 4) inverting the stereochemistry of polyketides by engineering the KR active sites and the interaction sites between KRs and ACPs.