Abstract：
Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3β-Hydroxysteroid dehydrogenase(3βHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3βHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3βHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3βHSD2 was 774 bp and encoded 257 residues. Dp3βHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3βHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3βHSD1 had stronger catalytic capacity than Dp3βHSD2. The expression level of Dp3βHSD1 was much higher than that of Dp3βHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3βHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.
摘要：
洋地黄毒素,洋地黄的重要次生代谢产物,是临床上常用的强心剂。3β-羟基类固醇脱氢酶（3βHSD）是参与洋地黄毒苷生物合成的关键酶。它属于短链脱氢酶/还原酶（SDR）家族，通过氧化和异构化前体甾醇在强心苷的生物合成中发挥作用。在这项研究中,从紫癜中克隆了两个3βHSD基因。结果表明,Dp3βHSD1的开放阅读框(ORF)为780bp,编码259个氨基酸残基。Dp3βHSD2的ORF为774bp,编码257个残基。Dp3βHSD1/2具有辅因子结合位点TGxxxA/GxG和催化位点YxxxK。体外实验证实Dp3βHSD1/2催化孕烯醇酮生成孕酮，Dp3βHSD1的催化能力强于Dp3βHSD2。叶片中Dp3βHSD1的表达水平远高于Dp3βHSD2,洋地黄毒苷只在树叶中积累。结果表明，Dp3βHSD1在洋地黄毒素的生物合成中在孕烯醇酮脱氢产生孕酮中起作用。本研究为进一步探索紫藤强心苷的生物合成途径提供了参考。
