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 stereospecifically hydroxylated by the cytochrome
P450 enzymes (CYP) Lactuca sativa costunolide synthase
CYP71BL2 (LsCOS) and Helianthus annuus GAA 8β-hydroxylase
CYP71BL1 (HaG8H) at C6 (in α-orientation) or C8 (in β-orientation),
respectively. Spontaneous subsequent lactonization of the resulting
6α-hydroxy-GAA leads to costunolide, whereas 8β-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
(8β-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 sunflowers,
using as a substrate 8β-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 sunflowers: 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.