Biosynthesis of γ-Alkylidenebutenolide Derivatives Reveals an Atom-Deleting Lactone Ring Contraction by Dual-Enzyme Cascade

Ring contraction represents a characteristic atom-level skeletal editing strategy in natural products. While this biosynthetic approach has garnered increasing attention, its role in modifying the lactone ring size remains underexplored. Here, we report a novel δ- to γ-lactone contraction during γ-alkylidenebutenolide biosynthesis in <i>Aspergillus candidus</i>. This dual-enzymatic cascade involves multifunctional oxidase PesD oxidizing a PKS-derived α-pyrone to a cyclic carbonate intermediate, followed by the DUF3237 enzyme PesF excising a molecule of CO₂ to achieve ring contraction─an unprecedented function for this enzyme family. Computational, crystallographic, and site-directed mutagenesis studies reveal that PesF employs a noncanonical acid-base mechanism, with Glu99 serving as a key catalytic residue that fulfills an unusual dual role.