Overexpression and RNAi-mediated Knockdown of Two 3 β-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis

Planta Med DOI: 10.1055/a-2074-91863β-hydroxy-Δ5-steroid dehydrogenases (3βHSDs) are supposed to be involved in 5β-cardenolide biosynthesis. Here, a novel 3βHSD (Dl3βHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3βHSD1 and Dl3βHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3βHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3βHSD1, Dl3βHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3βHSDs was realised by Agrobacterium-mediated transfer of Dl3βHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3βHSD1 and 35S:Dl3βHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3βHSD1 lines than in the controls. In the 35S:Dl3βHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH fo...
Source: Planta Medica - Category: Drugs & Pharmacology Authors: Tags: Original Papers Source Type: research