Molecular and enzymatic characterizations of novel bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases from Arabidopsis thaliana

We have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3 beta-hydroxysteroid dehydrogenase/C-4 decarboxylases (3 beta HSD/D) involved in sterol synthesis, termed At3 beta HSD/D1 and At3 beta HSD/D2. Transformation of the yeast ergosterol auxotroph erg26 mutant, which lacks 3 beta HSD/D activity, with the At3 beta HSD/D1 isoform or with At3 beta HSD/D2 isoform containing a C-terminal At3 beta HSD/D1 endoplasmic reticulum-retrieval sequence restored growth and ergosterol synthesis in erg26. An in vitro enzymatic assay revealed high 3 beta HSD/D activity for both isoenzymes in the corresponding microsomal extracts. The two At3 beta HSD/D isoenzymes showed similar substrate specificities that required free 3 beta-hydroxyl and C-4-carboxyl groups but were quite tolerant in terms of variations of the sterol nucleus and side chain structures. Data obtained with 4 alpha-carboxy-cholest-7-en-3 beta-ol and its 3 alpha-deuterated analog revealed that 3 alpha-hydrogen-carbon bond cleavage is not the rate-limiting step of the reaction. In planta reduction on the expression of the 3 beta HSD/D gene as a consequence of VIGS-mediated gene silencing in Nicotiana benthamiana led to a substantial accumulation of 3 beta-hydroxy-4 beta,14-dimethyl-5 alpha-ergosta-9 beta,19-cyclo-24(24(1))-en-4 alpha-carboxylic acid, consistent with a decrease in 3 beta HSD/D activity. These two novel oxidative decarboxylases constitute the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants.