Chisuga Taichi, Takinami Shota, Liao Zengwei, Karasawa Masayuki, Adachi Naruhiko, Kawasaki Masato, Moriya Toshio, Senda Toshiya, Terada Tohru, Kudo Fumitaka, Eguchi Tadashi, Nakano Shogo, Ito Sohei, Miyanaga Akimasa
Graduate School of Integrated Pharmaceutical and Nutritional Science, University of Shizuoka, Shizuoka, Japan.
Department of Chemistry, Tokyo Institute of Technology (Institute of Science Tokyo), Tokyo, Japan.
Nat Commun. 2025 Jul 25;16(1):6847. doi: 10.1038/s41467-025-62168-0.
Modular polyketide synthases (PKSs) are large multi-domain enzymes critical for the biosynthesis of polyketide antibiotics. However, challenges with structural analysis limits our mechanistic understanding of modular PKSs. In this report, we explore the potential of ancestral sequence reconstruction (ASR) for structure analysis of target proteins. As a model, we focus on the FD-891 PKS loading module composed of ketosynthase-like decarboxylase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains. We construct a KSAncAT chimeric didomain by replacing the native AT with an ancestral AT (AncAT) using ASR. After confirming that KSAncAT chimeric didomain retains similar enzymatic function to the native KSAT didomain, we successfully determine a high-resolution crystal structure of the KSAncAT chimeric didomain and cryo-EM structures of the KS-ACP complex. These cryo-EM structures, which could not be determined for the native protein, exemplify the utility of ASR to enable cryo-EM single-particle analysis. Our findings demonstrate that integrating ASR with structural analysis provides deeper mechanistic insight into modular PKSs. Furthermore, applying ASR to a partial region of the targeted multi-domain proteins could expand the potential of ASR and may serve as a valuable framework for investigating the structure and function of various multi-domain proteins.
模块化聚酮合酶（PKSs）是一类大型多结构域酶，对聚酮类抗生素的生物合成至关重要。然而，结构分析面临的挑战限制了我们对模块化PKSs作用机制的理解。在本报告中，我们探索了祖先序列重建（ASR）在目标蛋白结构分析中的潜力。作为模型，我们聚焦于由酮合成酶样脱羧酶（KS）、酰基转移酶（AT）和酰基载体蛋白（ACP）结构域组成的FD - 891 PKS加载模块。我们利用ASR，通过用祖先AT（AncAT）替换天然AT构建了KSAncAT嵌合双结构域。在确认KSAncAT嵌合双结构域保留了与天然KSAT双结构域相似的酶活性后，我们成功确定了KSAncAT嵌合双结构域的高分辨率晶体结构以及KS - ACP复合物的冷冻电镜结构。这些天然蛋白无法确定的冷冻电镜结构例证了ASR在实现冷冻电镜单颗粒分析方面的效用。我们的研究结果表明，将ASR与结构分析相结合能为模块化PKSs提供更深入的作用机制见解。此外，将ASR应用于靶向多结构域蛋白的部分区域可以拓展ASR的潜力，并可能为研究各种多结构域蛋白的结构和功能提供有价值的框架。
