The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and – according to a structural model – active site architecture are highly similar to those of the fusicocca‐2,10(14)‐diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6‐5‐5‐5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation‐reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site‐directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.

中文翻译：

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Asperfumene 和 Fusicoccadiene 合酶之间的功能转换以及通过邻位去质子化-再质子化过程进入 Asperfumene 生物合成


二萜合酶 AfAS 是从烟曲霉中鉴定出来的。它的氨基酸序列和（根据结构模型）活性位点结构与 fusicocca-2,10(14)-二烯合酶 PaFS 高度相似，但 AfAS 产生结构更加复杂的二萜，具有新型 6-5 ‐5‐5 四环骨架称为 asperfumene。通过同位素标记实验和DFT计算阐明了AfAS的环化机制。反应级联在其初始步骤中通过与 PaFS 级联类似的中间体进行，但随后通过不寻常的邻位去质子化-重质子化过程发生分歧，该过程在通向独特 asperfumene 骨架的步骤入口处触发骨架重排。结构模型仅揭示了活性位点之间的一个主要差异：PaFS 残基 F65 被 AfAS 中的 I65 取代。有趣的是，两种二萜合酶的定点诱变实验表明，与结构不太复杂的二萜相比，65 位作为四环 asperfumene 生物合成的双向功能开关。