Department of Chemistry, Haverford College, Haverford, PA, USA.
Methods Mol Biol. 2022;2489:239-267. doi: 10.1007/978-1-0716-2273-5_13.
The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs.
构成 II 型聚酮合酶 (PKS) 的酶是强大的生物催化剂，如果得到充分理解并加以战略应用，就能够以具有成本效益且可持续的方式获得一系列具有药用相关性的分子。实现这一目标的关键在于获得大量的材料，以便对这些合成酶的成分进行体外特性描述和结构分析。PKS 的一个核心组成部分是酰基载体蛋白 (ACP)，它在 II 型聚酮的生物合成过程中充当枢纽。在此，我们分享通过大肠杆菌中的异源表达来获取 II 型 PKS ACP 的方法。我们还分享了如何利用反应性和特异性光谱探针的安装来研究 II 型 PKS ACP 的构象动力学和相互作用。
