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<p>Macrocyclization is crucial in natural product biosynthesis for enhancing molecular rigidity and stability. Although thioesterase-mediated macrolactonization or macrolactamization is the predominant mechanism in type I polyketide synthases, here we report an alternative macrocyclization mechanism in which nuclear transport factor 2 (NTF2)-like enzymes catalyse a tandem stereoselective Michael addition and Knoevenagel condensation to construct a tetrahydrofuran-fused macrocyclic carbocycle. Genome mining identified a family of NTF2-like proteins that share this tandem cyclization capability. X-ray crystal structures complexed with substrate mimics and structure-based mutagenesis reveal that a lysine residue forms an iminium intermediate with the terminal aldehyde to enable cyclization, while an aspartic acid acts as a general base to mediate proton transfers. Structures capturing distinct states, from linear precursor to precyclization, provide direct insight into the ring-closure process. This work elucidates an iminium-catalysed tandem cyclization mechanism, expanding the known catalytic repertoire of NTF2-like enzymes and highlighting the potential of iminium-based biocatalysis in natural product biosynthesis.</p><p></p>

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Structural and mechanistic insights into iminium-catalysed macrocyclization by nuclear transport factor 2-like enzymes

  • 期刊论文

  • 发表日期：

    2026年1月29日

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  • Cheng Li Liu,
  • Bo Zhang,
  • Cheng Yuan Yuan,
  • Zi Jie Wang,
  • Ao Zhu,
  • Si Yuan Wang,
  • Zhang Yuan Yan,
  • Andrey Lebedev,
  • Yasuhiro Igarashi,
  • Ren Xiang Tan,
  • Hui Ming Ge

摘要

Macrocyclization is crucial in natural product biosynthesis for enhancing molecular rigidity and stability. Although thioesterase-mediated macrolactonization or macrolactamization is the predominant mechanism in type I polyketide synthases, here we report an alternative macrocyclization mechanism in which nuclear transport factor 2 (NTF2)-like enzymes catalyse a tandem stereoselective Michael addition and Knoevenagel condensation to construct a tetrahydrofuran-fused macrocyclic carbocycle. Genome mining identified a family of NTF2-like proteins that share this tandem cyclization capability. X-ray crystal structures complexed with substrate mimics and structure-based mutagenesis reveal that a lysine residue forms an iminium intermediate with the terminal aldehyde to enable cyclization, while an aspartic acid acts as a general base to mediate proton transfers. Structures capturing distinct states, from linear precursor to precyclization, provide direct insight into the ring-closure process. This work elucidates an iminium-catalysed tandem cyclization mechanism, expanding the known catalytic repertoire of NTF2-like enzymes and highlighting the potential of iminium-based biocatalysis in natural product biosynthesis.