Structural Insights into Three Sesquiterpene Synthases for the Biosynthesis of Tricyclic Sesquiterpenes and Chemical Space Expansion by Structure-Based Mutagenesis

The
cyclization of farnesyl diphosphate (FPP) into highly strained
polycyclic sesquiterpenes is challenging. We here determined the crystal
structures of three sesquiterpene synthases (STSs, namely, BcBOT2,
DbPROS, and CLM1) catalyzing the biosynthesis of the tricyclic sesquiterpenes
presilphiperfolan-8β-ol (1), Δ6-protoilludene (2), and longiborneol (3). All three STS structures contain a substrate mimic, the benzyltriethylammonium
cation (BTAC), in their active sites, providing ideal templates for
quantum mechanics/molecular mechanics (QM/MM) analyses toward their
catalytic mechanisms. The QM/MM-based molecular dynamics (MD) simulations
revealed the cascade reactions toward the enzyme products, and different
key active site residues that play important roles in stabilizing
reactive carbocation intermediates along the three pathways. Site-directed
mutagenesis experiments confirmed the roles of these key residues
and concomitantly resulted in 17 shunt products (4–20). Isotopic labeling experiments addressed the key hydride
and methyl migrations toward the main and several shunt products.
These combined methods provided deep insights into the catalytic mechanisms
of the three STSs and demonstrated how the chemical space of STSs
can rationally be expanded, which may facilitate applications in synthetic
biology approaches toward pharmaceutical and perfumery agents.