Received March 25, 2004
Returned for revision March 29, 2004
Accepted March 29, 2004
Novel Biosynthetic Pathway of Castasterone from Cholesterol in Tomato
Tae-Wuk Kim , Soo Chul Chang , June Seung Lee , Suguru Takatsuto , Takao Yokota , and Seong-Ki Kim *
Department of Life Science, Chung-Ang University, Seoul 156-756, Korea
University College, Yonsei University, Seoul 120-749, Korea
Department of Biological Science, Ewha Womans University, Seoul 120-750, Korea
Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943-8512, Japan
Department of Biosciences, Teikyo University, Utsunomiya 320-8551, Japan
* Corresponding author; email: skkimbio{at}cau.ac.kr.
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.
