Functional characterization of oxidosqualene cyclases and CYP716As associated with triterpene biosynthesis from...

Pentacyclic triterpenoids (PTs), characterized by their structural diversity, have been found to have therapeutic effects including anti-inflammatory, liver repair, neuroprotection. Corydalis yanhusuo used as an analgesic herbal remedy in the clinic, has pharmacologically active PT derivatives. β-hydroxy-olean-11,13(18)-dien-28-oic acid, as a validated PT in Corydalis yanhusuo, the detailed elucidation of its biosynthesis remains unclear. In this study, we successfully isolated two functional 2,3-oxidosqualene synthases (CyOSC1, CyOSC2) and one functional cytochrome P450 (CyCYP716A467) from Corydalis yanhusuo involved in its biosynthesis. Both CyOSC1 and CyOSC2 exclusively catalyze the cyclization of 2,3-oxidosqualene to produce β-amyrin, and then CyCYP716A467 oxidizes at the C-28 position of β-amyrin, yielding erythrodiol as the final product. With semi-rational protein engineering, 3 key residues (Gly62, Trp261 and Glu718) in CyOSC1 were found, which make sense in catalytic activity and mechanism. Dynamics simulations revealed that Gly62, despite not being located within the active pocket, could influence protein structural flexibility, affecting the substrate binding process and the overall reaction. This study provided a novel source of enzyme elements and lays the groundwork for unraveling the biosynthetic pathway of 3β-hydroxy-olean-11,13(18)-dien-28-oic acid, also identified unique residues that influenced the activity of CyOSC, contributed a new insight to protein engineering of OSCs.