Biosynthesis of eupatolide - a metabolic route for sesquiterpene lactone formation involving the P450 enzyme CYP71DD6.

Sesquiterpene lactones are a class of natural compounds well-known for their bioactivity and are characteristic for the Asteraceae family. Most sesquiterpene lactones are considered derivatives of germacrene A acid (GAA). GAA can be stereo-specifically hydroxylated by the cytochrome P450 enzymes (CYP) Lactuca sativa costunolide synthase CYP71BL2 (LsCOS) and Helianthus annuus GAA 8-hydroxylase CYP71BL1 (HaG8H) at C6 (in -orientation) or C8 (in -orientation), respectively. Spontaneous subsequent lactonization of the resulting 6-hydroxy-GAA leads to costunolide, whereas 8-hydroxy-GAA has not yet been reported to cyclize to a sesquiterpene lactone. Sunflower and related species of the Heliantheae tribe contain sesquiterpene lactones mainly derived from inunolide (7,8-cis lactone) and eupatolide (8-hydroxy-costunolide) precursors. However, the mechanism of 7,8-cis lactonization in general, and the 6,7-trans lactone formation in the sunflower tribe, remain elusive. Here we show that in plant cells, heterologous expression of CYP71BL1 leads to the formation of inunolide. Using a phylogenetic analysis of enzymes from the CYP71 family involved in sesquiterpenoid metabolism, we identified the CYP71DD6 gene which was able to catalyze the 6,7-trans lactonization in sunflower, using as substrate 8-hydroxy-GAA. Consequently, CYP71DD6 resulted in the synthesis of eupatolide, thus called HaES (Helianthus annuus eupatolide synthase). Thus, our study shows the entry point for the biosynthesis of two distinct types of sesquiterpene lactones in sunflower: the 6,7-trans lactones derived from eupatolide and the 7,8-cis lactones derived from inunolide. The implications for tissue-specific localization, based on expression studies are discussed.