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.