G2 Helix as the Plastic Region Modulating Diastereoselectivity of Cyclization in Fungal Bifunctional Sesterterpene Synthases

Sesterterpenoids are a rare class of terpenes with potential pharmaceutical applications. To date, most of the currently characterized fungal sesterterpenoids are produced through the catalysis of bifunctional sesterterpene synthases (BF-STPSs). However, the full catalytic potential of BF-STPSs is not yet fully understood. Here, using site-directed mutagenesis and computational simulations, we identified the G2 helix of BF-STPSs as a structurally plastic region that governs divergent cyclization pathways, leading to the formation of unique terpenes with distinct diastereoselectivity. Mutations in the functional site residues of the G2 helix in Type A BF-STPSs, DpPS and FoFS, altered the conformational flexibility of the carbocation intermediates, yielding a series of previously uncharacterized sesterterpenes with unprecedented diastereoselectivity in B-ring cyclization via a selective attack on the diastereotopic faces of the olefin. Notably, FoFS-V195A catalyzed the formation of five different skeleton types, while DpPS-V194A represented a rare example of a protein-engineered switch from a bicyclic to a tricyclic skeleton with a Z-configured Δ11,12 double bond. Density functional theory (DFT) calculations and molecular dynamics (MD) simulations provided further insights into the reaction details and active site dynamics. These findings highlight key amino acids within the G2 helix of BF-STPSs as mutational hotspots, providing an efficient strategy to accelerate the discovery of structurally diverse sesterterpenes.