Salvia divinorum (Lamiaceae) is an annual herb used by indigenous cultures of Mexico for medicinal and ritual purposes. The biosynthesis of salvinorin A, its major bioactive neo-clerodane diterpenoid, remains virtually unknown. This investigation aimed to identify the enzyme that catalyzes the first reaction of salvinorin A biosynthesis, the formation of (-)-kolavenyl diphosphate [(-)-KPP], which is subsequently dephosphorylated to afford (-)-kolavenol. Peltate glandular trichomes were identified as the major and perhaps exclusive site of salvinorin accumulation in S. divinorum. The trichome-specific transcriptome was used to identify candidate diterpene synthases (diTPSs). In vitro and in planta characterization of a class II diTPS designated as SdKPS confirmed its activity as (-)-KPP synthase and its involvement in salvinorin A biosynthesis. Mutation of a phenylalanine into histidine in the active site of SdKPS completely converts the product from (-)-KPP into ent-copalyl diphosphate. Structural elements were identified that mediate the natural formation of the neo-clerodane backbone by this enzyme and suggest how SdKPS and other diTPSs may have evolved from ent-copalyl diphosphate synthase.
Keywords: (–)-kolavenol; (–)-kolavenyl diphosphate; Salvia divinorum; class II diterpene synthase; diterpenoid diversification; neo-clerodane diterpenoid; neo-functionalization; product specificity; repeated evolution; salvinorin A biosynthesis.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.