Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Protein Sci. 2011 Jul;20(7):1244-55. doi: 10.1002/pro.652.
The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃-S-ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.
聚酮合酶（PKSs）的装配线结构为合理重编程聚酮生物合成途径以生产新型抗生素提供了机会。实现这一目标的一个基本挑战是确定控制这些酶装配线中反应性生物合成中间体单向通道化的因素。在每个 PKS 模块的催化循环中，酰基载体蛋白（ACP）首先与自身同源二聚体中配对亚基的酮合酶（KS）结构域合作，以延长生长中的聚酮链，然后与下一个模块的 KS 结构域合作，将新延长的聚酮链转移。我们使用 NMR 光谱研究了结构特征明确的 6-去氧赤藓醇 B 合酶 ACP 结构域的特征，这些特征有助于其与 KS 易位伙伴的结合。我们不仅能够可视化两个伙伴之间的选择性蛋白-蛋白相互作用，而且还检测到酰基链底物对这种相互作用的显著影响。开发了一种新型试剂 CF₃-S-ACP，作为蛋白质-蛋白质相互作用的 ¹⁹F NMR 光谱探针。讨论了我们的发现对理解模块间链易位的意义。
