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<p>Ergosteryl-3β-O-L-aspartate synthase (ErdS) catalyzes tRNA-dependent aspartylation of ergosterol, a lipid essential for fungal cell membrane integrity. However, the functional significance of ergosteryl-aspartate and the molecular mechanisms underlying its synthesis remain unclear. Here, we show that ErdS localization is highly dynamic and that Erg-Asp is required for proper hyphal growth, sporulation, and spore germination, and likely influences stress tolerance. The cryo-electron microscopy structure of ErdS revealed an unprecedented sterol-binding pocket. In addition, the structures in complex with a non-hydrolyzable Asp-N-tRNA<sup>Asp</sup> show a tRNA-guided intramolecular aminoacyl transfer mechanism between two functional domains of the enzyme. The CCA end of tRNA<sup>Asp</sup> undergoes a large displacement to reach the aa-tRNA transfer active site, while the tRNA elbow is clamped by a long extension an N-terminal α-helix. The present structural and mutational analyses demonstrate that domain fusion, dynamic repositioning, and tRNA-mediated substrate handover underlie the multifunctional catalytic efficiency of ErdS and participates in Erg-Asp synthesis independently from protein synthesis. These findings elucidate the regulatory mechanism of tRNA-dependent sterol modification and provide insights into fungal membrane dynamics, highlighting potential targets for antifungal therapies.</p>

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Structure of ergosteryl-aspartate synthase reveals how an entrapped tRNA is used like a prosthetic swinging arm in the synthesis of aminoacylated sterols

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月28日

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  • Hanako Murayama,
  • Nathaniel Yakobov,
  • Nassira Mahmoudi,
  • Sasha Legrosdidier,
  • Nicolas Fournier,
  • Solène Zuttion,
  • Kanata Matsumoto,
  • Michihiro Nishimura,
  • Jingwei Ji,
  • Bruno Senger,
  • Laurence Huck,
  • Howard B. Gamper,
  • Yoshiaki Kise,
  • Ralph E. Kleiner,
  • Mathieu Frechin,
  • Ya-Ming Hou,
  • Hubert D. Becker,
  • Yuzuru Itoh,
  • Frédéric Fischer,
  • Osamu Nureki

摘要

Ergosteryl-3β-O-L-aspartate synthase (ErdS) catalyzes tRNA-dependent aspartylation of ergosterol, a lipid essential for fungal cell membrane integrity. However, the functional significance of ergosteryl-aspartate and the molecular mechanisms underlying its synthesis remain unclear. Here, we show that ErdS localization is highly dynamic and that Erg-Asp is required for proper hyphal growth, sporulation, and spore germination, and likely influences stress tolerance. The cryo-electron microscopy structure of ErdS revealed an unprecedented sterol-binding pocket. In addition, the structures in complex with a non-hydrolyzable Asp-N-tRNAAsp show a tRNA-guided intramolecular aminoacyl transfer mechanism between two functional domains of the enzyme. The CCA end of tRNAAsp undergoes a large displacement to reach the aa-tRNA transfer active site, while the tRNA elbow is clamped by a long extension an N-terminal α-helix. The present structural and mutational analyses demonstrate that domain fusion, dynamic repositioning, and tRNA-mediated substrate handover underlie the multifunctional catalytic efficiency of ErdS and participates in Erg-Asp synthesis independently from protein synthesis. These findings elucidate the regulatory mechanism of tRNA-dependent sterol modification and provide insights into fungal membrane dynamics, highlighting potential targets for antifungal therapies.