Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

Triterpenoids are a large and medicinally important groupof naturalproducts with a wide range of biological and pharmacological effects.Among them, malabaricane-type triterpenoids are a rare group of terpenoidswith a 6,6,5-tricyclic ring system, and a few malabaricane triterpenesynthases have been characterized to date. Here, an arabidiol synthaseAmAS for the formation of the malabaricane-type 6,6,5-tricyclic triterpenoidskeleton in astramalabaricosides biosynthesis was characterized from Astragalus membranaceus. Multiple sequence alignment, site-directedmutagenesis, and molecular docking of AmAS reveal that residues Q256and Y258 are essential for AmAS activity, and the triad motif IIH725-727was the critical residue necessary for its product specificity. Mutationof IIH725-727 with VFN led to the formation of seven tricyclic, tetracyclic,and pentacyclic triterpenoids (1-7). Glycosylation of malabaricane-type triterpenoids in the biosynthesisof astramalabaricosides was also explored. Three triterpenoids (1, 5, and 6) displayed potent inhibitoryeffects against influenza A virus in vitro. These findings provideinsights into malabaricane-type triterpenoids biosynthesis in A. membranaceus and access to diverse bioactive triterpenoidsfor drug discovery.