Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC.

The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to 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 arrangement reaction, in which the carbocation formed from C-2 moves to the other side of the skeleton, runs back to C-3 to yield a friedelin cation, and finally deprotonated. However, as the data on the crystal structure of plant OSCs are lacking, it remains unknown why the CSR cascade reactions for friedelin biosynthesis and 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 yield. Furthermore, we semi-rationally designed two TwOSC mutants, which significantly improved the yields of friedelin and β-amyrin, respectively. Our findings provide novel insights into the CSR mechanism under TwOSC enzyme catalysis and propose a new strategy for the semi-rational design of other OSCs to promote PT biosynthesis.