酰基载体蛋白ACP变构驱动的酰基转移分子机制研究

Acyl carrier protein (ACP) carries acyl chains involved in dehydrogenation, hydration, hydrolysis, cyclization, acyl transfer and other enzyme-catalyzed reactions and is an important accessory protein in cells. Generally the selectivity of ACP-dependent enzymes for acyl chains is considered to be the catalytic properties of the enzyme, while ACP as a part of the substrate, the selectivity of the enzyme for acyl-ACP instead of acyl group only should be the catalytic properties of the enzyme. So the puzzle of partial acyl chain determination mechanism of substrate selection exists in many enzymes that catalyze acyl-ACP reactions. It is discovered in the study of acyltransferase GPAT by the applicant that the allosteric structure of ACP plays an important role in driving the acyl transfer process. However, the structural basis of its role remains unclear. This project is based on the scale synthesis of a series of acyl-ACP platform, using circular dichroism and X-ray diffraction to analyze the contribution of acyl chains and key amino acid residues to the structural changes of acyl-ACP; by the complex crystal structures of ACP and enzyme it will be obtained its static interaction model. And the dynamic visualization of fluorescent probes will provide the information of characterization during the interaction of acyl-ACP and GPAT. The catalytic mechanism of ACP's allosteric-driven acyl transfer will be revealed, which is the fundamental stone for providing series of ACP parts and rational synthesis strategies of the natural products synthesis such as fatty acids and polyketides.

酰基载体蛋白ACP携带酰基链参与脱氢、水解、环化及酰基转移等酶催化反应，是生命活动中重要的辅助蛋白。长期以来，酰基-ACP依赖型的相关酶对酰基链的选择被认为是该酶的催化特性，忽视了属于部分底物结构易变的ACP的影响，而对酰基-ACP整体的选择性才是该酶的催化特性。以酰基链部分确定底物选择性的误区存在于很多相关酶研究中。申请人在酰基转移酶GPAT的研究中发现，ACP的变构在驱动酰基转移过程中起重要的作用，然而其驱动反应的结构基础仍不清楚。本项目基于规模合成系列酰基-ACP的平台，利用圆二色谱和X射线衍射解析酰基链及关键氨基酸残基对酰基-ACP结构变化的贡献；在此基础上通过ACP和酶复合物晶体结构的单一状态，结合荧光探针动态可视化的表征酰基-ACP同GPAT作用过程的信息，揭示变构的酰基-ACP对酰基转移酶催化反应的分子机制，为脂肪酸、聚酮等天然或非天然产物合成提供系列ACP元件及新策略。

ACP 作为acyl-ACP的一部分作为反应的底物，而研究互作酶催化特性时往往只考虑酰基链却忽视了ACP部分。实际上互作酶识别的是整体acyl-ACP 的结构。ACP 由于其灵活多变的结构，与不同酰基相连，表现出结构不同，进而影响互作酶的性质。因此单从酰基链与互作酶的结构无法理解互作酶对酰基的选择性，这种选择机制的误解存在于很多催化 ACP 参与反应的酶中。本研究提出通过改变ACP而影响acyl-ACP的结构，进而改变互作酶的选择性。在以硫酯酶ChFatB2作为互作酶，实现了以EcACP为酰基载体时，ChFatB2偏好C10-EcACP，而对ClACP2 时，偏好C8-ClACP2。本研究利用分子动力学模拟和体外酶活分析揭示了MiGPAT1和ACP的识别界面，MiGPAT1上的K204和R212分别与ACP上的E57和I54发生相互作用。利用圆二色光谱、蛋白质芯片技术研究了不同acyl-ACP对硫酯酶ChFatB2催化特性的影响。结果表明相对于EcACP来说ClACP2与ChFatB2相互作用更强，ChFatB2更偏好ClACP2。不同酰基载体蛋白ACP直接影响硫酯酶链长选择性，以EcACP为酰基载体时，ChFatB2偏好C10-EcACP，以ClACP2为酰基载体时，ChFatB2偏好C8-ClACP2。酰基载体蛋白ACP的结构直接会影响acyl-ACP的结构从而导致与ChFatB2的识别发生变化，导致了互作酶的链长选择性的变化。这打破了硫酯酶调控脂肪酸链长的一贯认识，对合成生物学找到合适的硫酯酶与酰基载体蛋白ACP匹配以达到调控脂肪酸链长的目的提供了新的思路。

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