Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522–542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., Chem. Biol. 17, 776–785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein–protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528–547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein–protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

多步代谢途径中的酶利用一系列调节机制来维持微妙的体内平衡 [K. Magnuson, S. Jackowski, CO Rock, JE Cronan, Jr, Microbiol。Rev. 57, 522–542 (1993)]。载体蛋白尤其在代谢途径中适当酶之间穿梭底物方面发挥重要作用。虽然假设 [E. Płoskoń 等人，化学。生物。17, 776–785 (2010)]，对于任何酰基载体蛋白 (ACP) 依赖性途径，底物传递的变构调节从未被证明过。由于蛋白质-蛋白质相互作用的瞬时性和动态性、底物的多样性和底物的不稳定性，研究这些机制仍然具有挑战性[K. Finzel, DJ Lee, MD Burkart, ChemBioChem16, 528–547 (2015)]。在这里，我们展示了 ACP 和伙伴酶之间独特的通讯机制，使用溶液核磁共振光谱和分子动力学来阐明依赖于脂肪酸链长度的变构。我们证明伙伴酶可以通过蛋白质 - 蛋白质相互作用以变构方式区分链长，因为底物螯合的结构特征从 ACP 四螺旋束内转换到蛋白质表面，而无需随机链翻转。这些结果阐明了 ACP 货物通信的细节，可以作为工程载体蛋白质依赖性途径的基础，用于特定的所需产品。