钙霉素聚酮链释放亚基蛋白性质优化及底物选择性

Property optimization and substrate selectivity of a polyketide chain-releasing subunit CalA3 in calcimycin synthesis

摘要:

【目的】钙霉素合成酶亚基CalA3释放钙霉素合成过程中的聚酮链。获得生物化学性质稳定、蛋白结构性质均一的CalA3蛋白，可用于冷冻电镜(cryo-electron microscopy)结构解析，以帮助理解装配线型聚酮合酶亚基释放聚酮链的生物化学机理。探究CalA3对不同关键结构特征的聚酮链底物的选择性，可为制备CalA3与小分子化合物的复合物提供生化材料，同时也为进一步挖掘CalA3的成酰胺键的应用潜能提供借鉴。【方法】优化CalA3蛋白异源表达菌株的培养条件、CalA3蛋白纯化的生化条件，利用负染电镜观察蛋白形态，计算并分析蛋白质结构的性质；测定CalA3对不同结构的直链聚酮类似物的体外催化活性，利用色谱和质谱分析鉴定CalA3催化N-乙酰半胱氨酸-吡咯-2-丙酸(SNAC-C3)、N-乙酰半胱氨酸-戊酸(SNAC-C5)和月桂酰辅酶A等多种不同结构的直链聚酮底物类似物与3-羟基邻氨基苯甲酸(3-hydroxy anthranilic acid,3HA)反应的产物。【结果】利用优化后的培养基PGTY，不仅实现了高纯度巨型聚酮合酶CalA3超量异源表达，同时，负染电镜观察、计算分析表明蛋白颗粒的结构性质优异。使用该条件纯化获得的CalA3蛋白，可以用于制备冷冻电镜样品，进行结构解析。CalA3催化SNAC-C3、SNAC-C5与3HA发生氨解反应，未检测到CalA3对月桂酰辅酶A底物的催化活性。【结论】蛋白纯化和负染电镜结果显示，本研究成功建立了CalA3异源表达的大肠杆菌培养条件、蛋白纯化生化条件。体外催化实验结果表明CalA3对聚酮链底物的结构选择具有宽泛性。这些发现为进一步探究聚酮合酶的结构、功能及应用提供了一定的借鉴。

Abstract:

[Objective] CalA3 subunit catalyzes the release of the polyketide chain in the biosynthesis of calcimycin. This study aims to obtain CalA3 proteins with stable biochemical properties and homogeneous structure for cryo-electron microscopy, to understand the mechanism of polyketide chain releasing by the module of the polyketide synthase, and to explore the selectivity of CalA3 to polyketide substrates with different structures, thereby providing biochemical materials for the complexes of CalA3 with small ligands and a reference for further exploring the catalytic potential of CalA3. [Methods] The medium for culturing the heterologous expression strain and the biochemical conditions for CalA3 protein purification were optimized. Then, the CalA3 proteins were analyzed by negative-stain transmission electron microscopy. The reaction products of S-(2-acetamidoethyl)3-(1H-pyrrol-2-yl) propanethioate (SANC-C3), N-(2-(pentylthio) ethyl) acetamide (SNAC-C5), lauroyl-CoA and 3-hydroxy anthranilic acid (3HA) catalyzed by CalA3 were identified by high performance liquid chromatography and high-resolution mass spectrometry (LC-MS). [Results] The strains cultured in the optimized medium PGTY, expressed more CalA3 proteins which exhibited featured structures under negative-stain transmission electron microscope. The purified CalA3 proteins were prepared for cryo-electron microscopy for structural determination. CalA3 catalyzed the aminolysis reaction of SNAC-C5 and SNAC-C3 with 3HA. However, the products of lauroyl-CoA and 3HA catalyzed by CalA3 were not detected. [Conclusion] The results of protein purification and negative-stain transmission electron micrographs show that the conditions for culturing Escherichia coli for heterologous expression and purifying CalA3 proteins are established. The results of in vitro catalytic experiments demonstrate that CalA3 has broad selectivity for the structure of polyketide substrates. These findings provide a reference for further exploring the structure, functions, and application of polyketide synthases.