A methionine-lined active site governs carbocation stabilization and product specificity in a bacterial terpene synthase

Terpene synthases (TPSs) generate complex hydrocarbon scaffolds through carbocationic cyclization cascades that demand precise active-site control to stabilize reactive intermediates. While π–cation and electrostatic interactions are established stabilizing factors, the role of methionine has remained unclear. Here, we identify a methionine-rich active site in hydropyrene synthase (HpS), a bacterial Class I TPS involved in pseudopterosin biosynthesis. Crystallography, mutagenesis, and multiscale QM/MM simulations reveal that methionine residues provide steric guidance and direct sulfur–carbocation stabilization during catalysis. Mutations alter product distributions, confirming functional relevance. Quantum chemical calculations indicate that sulfur–carbocation interactions are energetically comparable to π–carbocation interactions. These results uncover a previously unrecognized mechanism of carbocation stabilization in terpene biosynthesis.