Fungal P450 Deconstructs the 2,5-Diazabicyclo[2.2.2]octane Ring En Route to the Complete Biosynthesis of 21R-Citrinadin A.

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.

An NmrA-like enzyme-catalysed redox-mediated Diels-Alder cycloaddition with anti-selectivity.

The Diels-Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels-Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels-Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions. Here we report the discovery and characterization of CtdP, a member of a new class of bifunctional oxidoreductase/Diels-Alderase, which was previously annotated as an NmrA-like transcriptional regulator. We demonstrate that CtdP catalyses the inherently disfavoured cycloaddition to form the bicyclo[2.2.2]diazaoctane scaffold with a strict α-anti-selectivity. Guided by computational studies, we reveal a NADP+/NADPH-dependent redox mechanism for the CtdP-catalysed inverse electron demand Diels-Alder cycloaddition, which serves as the first example of a bifunctional Diels-Alderase that utilizes this mechanism.