Exploring the Diverse Landscape of Fungal Cytochrome P450‐Catalyzed Regio‐ and Stereoselective Dimerization of Diketopiperazines

Abstract Dimeric indole‐containing diketopiperazines (di‐DKPs) are a diverse group of natural products produced through cytochrome P450‐catalyzed C–C or C–N coupling reactions. The regio‐ and stereoselectivity of these reactions plays a significant role in the structural diversity of di‐DKPs. Despite their pivotal role, the mechanisms governing the selectivity in fungi are not fully understood. Employing bioinformatics analysis and heterologous expression experiments, five undescribed P450 enzymes (AmiP450, AcrP450, AtP450, AcP450, and AtuP450) responsible for the regio‐ and stereoselective dimerization of diketopiperazines (DKPs) in fungi are identified. The function of these P450s is consistent with phylogenetic analysis, highlighting their dominant role in controlling the dimerization modes. Combinatorial biosynthesis‐based pathway reconstitution of non‐native gene clusters expands the chemical space of fungal di‐DKPs and reveals that the regioselectivity is influenced by the substrate. Furthermore, multiple sequence alignment and molecular docking of these enzymes demonstrate a C‐terminal variable region near the substrate tunnel entrance in AtuP450 that is crucial for its regioselectivity. These findings not only reveal the secret of fungal di‐DKPs diversity but also deepen understanding of the mechanisms and catalytic specificity involved in P450‐catalyzed dimerization reactions.