title: Novel biosynthetic pathway of castasterone from cholesterol in tomato.
abstract: Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs. The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol --> cholestanol and 6-deoxo-28-norteasterone <--> 6-deoxo-28-nor-3-dehydroteasterone <--> 6-deoxo-28-nortyphasterol --> 6-deoxo-28-norcastasterone --> 28-norcastasterone (28-norCS). Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS. The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85. Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant. These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS. Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM. Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [2H6]CS to [2H3]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [2H6]CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta. Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol. In addition, CS and [2H6]CS were not converted into BL and [2H6]BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS. In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings.
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Tae-Wuk
authors.initials: TW
authors.lastname: Kim
authors.forename: Soo Chul
authors.initials: SC
authors.lastname: Chang
authors.forename: June Seung
authors.initials: JS
authors.lastname: Lee
authors.forename: Suguru
authors.initials: S
authors.lastname: Takatsuto
authors.forename: Takao
authors.initials: T
authors.lastname: Yokota
authors.forename: Seong-Ki
authors.initials: SK
authors.lastname: Kim
mesh_terms: D002777:Cholestanols / Q000737:chemistry / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism*
chemical_list: D002777:Cholestanols; C083545:castasterone; D002784:Cholesterol