Modeling the Impact of Active Site Mutations on the Quenching Fate and Cyclization Depth of a Rare Oxidosqualene Cyclase

More importantly, we demonstrate that mechanism-guided active site substitutions can dramatically alter the chemical trajectory. Relaxing steric constraints (F728A) induces catalytic promiscuity to yield tricyclic and tetracyclic products. Furthermore, the L259Y mutation significantly alters the quenching network.

It unlocks the hydroxylation pathway to form dammarenediol II and acts as an engineered base extending cyclization depth to yield the pentacyclic lupeol. Notably, combining these functional loci (L259Y+F728S) reveals notable synergistic epistasis between the steric cavity and the proton-transfer network, driving competitive hydration events to diversify the hydroxylated product profile. This study provides a comprehensive mechanistic framework for understanding how OSC active site mutations govern product specificity, offering a robust theoretical basis for the tailored, mechanism-driven functional reprogramming of triterpene synthases in synthetic biology.