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Ancestral sequence reconstruction as a tool for structural analysis of modular polyketide synthases

Author

Listed:
  • Taichi Chisuga

    (University of Shizuoka
    Tokyo Institute of Technology (Institute of Science Tokyo))

  • Shota Takinami

    (University of Shizuoka)

  • Zengwei Liao

    (The University of Tokyo
    University of California at Berkeley)

  • Masayuki Karasawa

    (The University of Tokyo)

  • Naruhiko Adachi

    (High Energy Accelerator Research Organization (KEK)
    University of Tsukuba)

  • Masato Kawasaki

    (High Energy Accelerator Research Organization (KEK))

  • Toshio Moriya

    (High Energy Accelerator Research Organization (KEK))

  • Toshiya Senda

    (High Energy Accelerator Research Organization (KEK))

  • Tohru Terada

    (The University of Tokyo
    The University of Tokyo)

  • Fumitaka Kudo

    (Tokyo Institute of Technology (Institute of Science Tokyo)
    Kanagawa University)

  • Tadashi Eguchi

    (Tokyo Institute of Technology (Institute of Science Tokyo))

  • Shogo Nakano

    (University of Shizuoka
    Japan Science and Technology Agency)

  • Sohei Ito

    (University of Shizuoka)

  • Akimasa Miyanaga

    (Tokyo Institute of Technology (Institute of Science Tokyo)
    The University of Tokyo
    The University of Tokyo)

Abstract

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 (KSQ), acyltransferase (AT) and acyl carrier protein (ACP) domains. We construct a KSQAncAT chimeric didomain by replacing the native AT with an ancestral AT (AncAT) using ASR. After confirming that KSQAncAT chimeric didomain retains similar enzymatic function to the native KSQAT didomain, we successfully determine a high-resolution crystal structure of the KSQAncAT chimeric didomain and cryo-EM structures of the KSQ–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.

Suggested Citation

  • Taichi Chisuga & Shota Takinami & Zengwei Liao & Masayuki Karasawa & Naruhiko Adachi & Masato Kawasaki & Toshio Moriya & Toshiya Senda & Tohru Terada & Fumitaka Kudo & Tadashi Eguchi & Shogo Nakano & , 2025. "Ancestral sequence reconstruction as a tool for structural analysis of modular polyketide synthases," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-62168-0
    DOI: 10.1038/s41467-025-62168-0
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