Engineering the D‑Helix in Fungal Sesterterpene Synthase ZbSS Unlocks a Skeletal Rearrangement Cyclization Route toward Sesterterpene Diversification

Skeletal rearrangement serves as a fundamental mechanism for generating sesterterpenoid structural diversity, yet the enzymatic control of these processes remains poorly understood. Here, by integrating AlphaFold2-predicted structural models with site-directed mutagenesis, we identify a single residue (Phe92/Ile92) on the D-helix of fungal sesterterpene synthase ZbSS that acts as a master modulator of skeletal rearrangement. The I92F mutation in ZbSS effectively reprogrammed its outcome, unlocking a rearrangement to produce the pentacyclic dia-quiannulatenes, a mechanism supported by prior DFT calculations. This represents the successful rational reprogramming of skeletal rearrangement in fungal bifunctional terpene synthases via a single residue. MD simulations further suggested that Phe92 steers the carbocation fate via stabilizing cation−π interactions. Our approach establishes a strategy for expanding terpene diversity through engineered skeletal rearrangements.