Identification of a 3β-Hydroxysteroid Dehydrogenase/3-Ketosteroid Reductase Involved in α-Tomatine Biosynthesis in Tomato

alpha-Tomatine and dehydrotomatine are major steroidal glycoalkaloids ( SGAs) that accumulate in the mature green fruits, leaves and flowers of tomato ( Solanum lycopersicum), and function as defensive compounds against bacteria, fungi, insects and animals. The aglycone of dehydrotomatine is dehydrotomatidine ( 5,6-dehydrogenated tomatidine, having the Delta(5,6) double bond; the dehydro-type). The aglycone of alpha-tomatine is tomatidine ( having a single bond between C5 and C6; the dihydro-type), which is believed to be derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 reduction and C3 reduction; however, these conversion processes remain uncharacterized. In the present study, we demonstrate that a short-chain alcohol dehydrogenase/reductase designated Sl3 beta HSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3 beta HSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of alpha-tomatine are accumulated. Biochemical analysis of the recombinant Sl3 beta HSD1 protein revealed that Sl3 beta HSD catalyzes the C3 oxidation of dehydrotomatidine to form tomatid-4-en-3-one and also catalyzes the NADH-dependent C3 reduction of a 3-ketosteroid ( tomatid-3-one) to form tomatidine. Furthermore, during co-incubation of Sl3 beta HSD with SlS5 alpha R1 ( steroid 5 alpha-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3 beta HSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in alpha-tomatine content. Furthermore, the constitutive expression of Sl3 beta HSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3 beta HSD is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in alpha-tomatine biosynthesis.