Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC

Abstract The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate ( 3S )‐2,3‐oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine‐step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and β ‐amyrin, respectively.