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A capillary GC-MS analysis revealed that two stereoisomers of castasterone are contained in immature seeds of Phaseolus vulgaris. 400 MHz proton NMR analysis of the stereoisomers determined they are A ring epimers of castasterone, 3-epicastasterone and 2,3-diepicastasterone. In rice lamina inclination assay, 3-epicastasterone and 2,3-diepicastasterone showed reduced biological activity than that of castasterone. Together with our previous finding that 2-epicastasterone exhibits a less biological activity than CS, this result indicates that epimerization of hydroxyl at C-2 or/and C-3 is/are inactive processes to reduce biological activity of CS after exerting as a bioactive brassinosteroid in P. vulgaris.

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There are over forty members of the class of compounds known as plant steroidal hormones.1-2 They are collectively named brassinosteroids (BRs). Among these, castasterone (CS) and brassinolide (BL) are considered the most important BRs because of their strong biological activities and wide-distribution in the plant kingdom. CS is biosynthesized from 24-methylcholesterol via two parallel pathways, namely, the early and late C-6 oxidation pathways, and then CS is further oxidized by 7-oxalactonation to produce BL. In the late C-6 oxidation pathway (Fig. 1), conversion of 6-deoxoCS to CS is catalyzed by two BR 6-oxidases which are cytochrome P450s designated as CYP85A1 and A2. 3 Recently we found that CYP85A2, has additional effects on BL synthase-it mediates the conversion of CS to BL, indicating that CYP85A2 is a bi-functional enzyme for BL synthase as well as for BR 6-oxidase. 4 As shown in Fig. 1, accumulating evidence suggests that plants operate multiple biosynthetic pathways to synthesize diverse naturally-occurring BRs such as 28-norBRs (C27-BRs), 24-methylene-BRs (C 28 -BRs), 24-methyl-BRs (C 28 -BRs) and 24-ethyl-BRs (C 29 -BRs).5 Although carbon skeletons, especially the alkyl groups at C-24, are different in C27-, C28-and C29-BR biosynthesis, biosynthetic reactions involved in the multiple BR biosyntheses are thought to be basically identical, suggesting that the same genes or proteins characterized in 24-methyl-BR biosynthesis are likely to catalyze the corresponding biosynthetic reactions for the other BR biosyntheses. 3 To test the possibility, involvement of CYP85A1 and A2, which are potentially useful proteins for enhancing BRs activity to develop commercially valuable plants, in 28-nor-, 24-methylene-and 24-ethyl-BRs biosynthesis was investigated in this study.The preparation of Cyt P450 overexpressing yeast strains (CYP85A1/V60/WAT21 and CYP85A2/V60/WAT21) and galactose induction were done as previously described.6 Galactose-induced cells were diluted in 20 mL of YPL to an OD 550 of 0.4 to 0.6 and about 5 ug of suitable substrates were added to the cells. After 6 hrs, metabolites were extracted using 20 mL of ethyl acetate and then subjected to reversed-phase HPLC (RP-HPLC) (SenshuPak C 18 , 10 × 150 mm) with a flow rate of 2.5 mL/min and 45% acetonitrile as the elution. Fractions were collected every minute. Fractions corresponding to the retention times of authentic BRs for expected products (28-norBL, DL: 10 min; 28-norCS, DS, BL: 14 min; 28-HBL: 16 min; CS: 20 min) were collected and analyzed by GC-MS after suitable derivatizations. 4 First, successful expressions of CYP85A1 and A2 in yeast strains (CYP85A1/V60/WAT21 and CYP85A2/V60/WAT21) were examined. When 6-deoxo-CS was added as a substrate, as summarized Table 1, the expressed CYP85A1 and CYP85A2 catalyzed the conversion of 6-deoxo-CS to CS. When CS was used as a substrate, the expressed CYP85A2 induced production of BL, but CYP85A1 did not. Therefore, it is confirmed that the function of the expressed CYP85A1 and A2 is correct.Second, a...