Localization and in-Vivo Characterization of Thapsia garganica CYP76AE2 Indicates a Role in Thapsigargin Biosynthesis

Abstract

The Mediterranean plant (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed in , converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis.

Comparative functional genomics analysis of cytochrome P450 gene superfamily in wheat and maize

“…Flavonoid 3′-hydroxylases (F3'Hs) and flavonoid 3′,5′-hydroxylases (F3'5'Hs) competitively control the synthesis of the precursors of blue and red anthocyanins, and CYP75B and CYP75A respectively function as F3'Hs and F3'5'Hs in the determination of flower colour [5]. Currently, Thapsigargin become a research hotspot of the anticancer drug Mipsagargin, and it is TenctlyThapsia garganica CYP76AE2 that mediates the conversion of epikunzeaol to epidihydrocostunolide compounds which are possible intermediates in thapsigargin biosynthesis [6]. Another essential role of CYP450s is the regulation of plant hormone homeostasis.…”

Discovery of a non‐stereoselective cytochrome P450 catalyzing either 8α‐ or 8β‐hydroxylation of germacrene A acid from the Chinese medicinal plant, Inula hupehensis

“…However, curiously the germacrene A‐type 12,8β‐lactones are formed in nature (Paknikar and Sardesai, ), and how such lactone ring can be formed remains unknown. Recently, the mechanism for forming a 12,6β‐lactone at the germacrene stage in thapsigargin biosynthesis has been described (Andersen et al ., ), in which a 6β‐hydroxyl group is already present prior to the successive oxidation on the C12, and then the C12 triple oxidation leads to the spontaneous formation of a 12,6β‐lactone ring. Therefore, similarly, to form the 12,8β‐lactone ring, it would be possible that the 8β‐hydroxylation on germacrene A might occur before the C12 triple oxidation by the GAO, as the enzyme GAO catalyzing the C12 triple oxidation has a broad substrate specificity (Gavira et al ., ; Eljounaidi et al ., ; Takase et al ., ), and may accommodate the 8β‐hydroxyl germacrene A as the substrate to facilitate the 12,8β‐lactone ring formation.…”

Traps and Pitfalls—Unspecific Reactions in Metabolic Engineering of Sesquiterpenoid Pathways

“…Examples for this are costunolide (1), inunolide (8) and its derivatives (exocyclic methylene group: double bond position ∆11→13). On the other hand, the inplanta production of epi-dihydrocostunolide (6), an STL without an exocyclic methylene group, did not result in cysteine or GSH adducts, which would support a nonenzymatic Michael-type addition [29]. This nucleophilic addition reaction of an α-methylene-γ-lactone with the thiol group of biomolecules such as cysteine has been known for a long time [34] as one of the main reasons for STL bioactivity.…”

“…The production of artemisinic acid (4) in Nicotiana benthamiana (Figure 1a) has been observed to yield mostly artemisinic acid 12-β-glucoside (5), which can be explained by (c) an esterification of the acid moiety of artemisinic acid to diglucose [28]. In planta produced epi-kunzeaol (6) was linked to two glucose units [29]. This was due to (d) an etherification of the C7-hydroxy moiety of epi-kunzeaol to form epi-kunzeaol-diglucoside (7).…”