Structural insights into γ-presodorifen pyrophosphate synthase

Canonical terpenoids conform to the (C₅H₈)_n formula, whereas an emerging group of terpenoids that break the isoprene rule have been discovered recently. The precursor of C₁₇ homosesquiterpenoid chlororaphens is derived from converting canonical C₁₅ farnesyl pyrophosphate (FPP) by two consecutive methyltransferases (MTs). The first step is catalyzed by a bi-functional S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates C10 on FPP to trigger a cyclization cascade and generate the monocyclic C₁₆ γ-presodorifen pyrophosphate (γ-PSPP). Here, we report the crystal structures of γ-PSPP synthase from Variovorax boronicumulans PHE5-4 (VbFPPMT) and Pseudomonas chlororaphis O6 (PcFPPMT), as well as the Mg²⁺ ion- and geranyl pyrophosphate (GPP)-bound complex of PcFPPMT. These structures reveal the overall structure of γ-PSPP synthases, composition of the catalytic center, and a flexible region responsible for the anchoring of the pyrophosphate (PPi) moiety of the substrate and a Mg²⁺ ion. Combining structural analysis, molecular docking, and site-directed mutagenesis, we identified key residues that govern the catalytic mechanism of these bi-functional γ-PSPP synthases. These findings advance our understanding into the catalytic mechanism employed by non-canonical methyltransferases, establish a vital framework for further mechanistic understanding and benefit rational engineering of C₁₆/C₁₇ terpenoid biosynthesis.