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.