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<p><i>C</i>-prenylated flavonoids possess notable pharmaceutical potential, but their production is hindered by the challenging selective prenylation of their unstable polyphenolic cores. Natural prenyltransferases offer a direct route but suffer from low activity and incomplete mechanistic understanding. Here, we report a directed evolution strategy to reshape the active pocket of the prenyltransferase AtaPT, uncovering an aromatic cage that governs both regioselectivity and donor specificity. By tuning cage occupancy, we engineer three mutants with high chemo- and regioselectivity toward dimethylallyl diphosphate or geranyl pyrophosphate. Structural analysis and molecular simulations validate the role of the cage in guiding flavonoid prenylation. Notably, the aromatic cage mechanism observed in AtaPT is not unique and can be recapitulated in homologous enzymes. Introduction of the aromatic cage consistently enhances both activity and selectivity, confirming its crucial role. AtaPT mutants enable the efficient and scalable synthesis of 27 <i>C</i>-prenylated flavonoids, including 8 previously unreported compounds. With an integrated donor regeneration system, preparative-scale biotransformations achieve product titers up to 400 mg/L. This study establishes a selective and scalable biocatalytic platform for flavonoid prenylation and offers mechanistic insights for enzyme engineering.</p>

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Tuning aromatic cage occupancy in prenyltransferases enables selective and efficient production of rare c-prenylated flavonoids

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月19日

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  • Ruiying Qiu,
  • Huisi Huang,
  • Junxi Chi,
  • Longwei Gao,
  • Qilin Gao,
  • Min Li,
  • Menghao Cai,
  • Haishuang Yu,
  • Shijie Wang,
  • Zhilan Qian,
  • Minchuan Jiang,
  • Yu Liu,
  • Bo Chen,
  • Yang Zhou,
  • Zhi-Min Zhang,
  • Jian-bo Wang

摘要

C-prenylated flavonoids possess notable pharmaceutical potential, but their production is hindered by the challenging selective prenylation of their unstable polyphenolic cores. Natural prenyltransferases offer a direct route but suffer from low activity and incomplete mechanistic understanding. Here, we report a directed evolution strategy to reshape the active pocket of the prenyltransferase AtaPT, uncovering an aromatic cage that governs both regioselectivity and donor specificity. By tuning cage occupancy, we engineer three mutants with high chemo- and regioselectivity toward dimethylallyl diphosphate or geranyl pyrophosphate. Structural analysis and molecular simulations validate the role of the cage in guiding flavonoid prenylation. Notably, the aromatic cage mechanism observed in AtaPT is not unique and can be recapitulated in homologous enzymes. Introduction of the aromatic cage consistently enhances both activity and selectivity, confirming its crucial role. AtaPT mutants enable the efficient and scalable synthesis of 27 C-prenylated flavonoids, including 8 previously unreported compounds. With an integrated donor regeneration system, preparative-scale biotransformations achieve product titers up to 400 mg/L. This study establishes a selective and scalable biocatalytic platform for flavonoid prenylation and offers mechanistic insights for enzyme engineering.