Prenylated indole alkaloids (PIAs) possess great structural diversity and show biological activities. Despite significant efforts in investigating the biosynthetic mechanism, the key step in the transformation of 2,5-diazabicyclo[2.2.2]octane-containing PIAs into a distinct class of pentacyclic compounds remains unknown. Here, using a combination of gene deletion, heterologous expression, and biochemical characterization, we show that a unique fungal P450 enzyme CtdY catalyzes the cleavage of the amide bond in the 2,5-diazabicyclo[2.2.2]octane system, followed by a decarboxylation step to form the 6/5/5/6/6 pentacyclic ring in 21R-citrinadin A. We also demonstrate the function of a subsequent cascade of stereospecific oxygenases to further modify the 6/5/5/6/6 pentacyclic intermediate en route to the complete 21R-citrinadin A biosynthesis. Our findings reveal a key enzyme CtdY for the pathway divergence in the biosynthesis of PIAs and uncover the complex late-stage post-translational modifications in 21R-citrinadin A biosynthesis.

中文翻译：

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真菌 P450 在完成 21R-Citrinadin A 生物合成的过程中解构 2,5-二氮杂双环[2.2.2]辛烷环

异戊二烯化吲哚生物碱（PIA）具有巨大的结构多样性并表现出生物活性。尽管在研究生物合成机制方面付出了巨大的努力，但将含有 2,5-二氮杂双环[2.2.2]辛烷的 PIA 转化为一类独特的五环化合物的关键步骤仍然未知。在这里，结合基因删除、异源表达和生化表征，我们证明了一种独特的真菌 P450 酶 CtdY 催化 2,5-二氮杂双环[2.2.2]辛烷系统中酰胺键的裂解，随后脱羧步骤，在 21 R -citrinadin A中形成 6/5/5/6/6 五环。我们还演示了随后的立体特异性加氧酶级联的功能，以进一步修饰 6/5/5/6/6 五环中间体途中完成 21 R -citrinadin A 生物合成。我们的研究结果揭示了 PIA 生物合成途径分歧的关键酶 CtdY，并揭示了 21 R -citrinadin A 生物合成中复杂的后期翻译后修饰。