洋地黄毒苷合成途径3β-羟基类固醇脱氢酶基因克隆及功能分析

Cloning and functional analysis of 3β-hydroxysteroid dehydrogenase gene involved in biosynthesis of digitoxin

洋地黄毒苷作为中药洋地黄中重要的次生代谢产物,是临床中最常用的强心药物之一。3β-羟基类固醇脱氢酶(3βHSD)是洋地黄毒苷生物合成途径酶,属于短链脱氢酶/还原酶(SDR)家族,起到氧化和异构前体化合物甾醇的作用,参与强心苷类化合物的生物合成。该研究从洋地黄中首次克隆得到2个3βHSD基因,实验结果显示:Dp3βHSD1开放阅读框为780 bp,编码259个氨基酸;Dp3βHSD2开放阅读框为774 bp,编码257个氨基酸。Dp3βHSD1/2均具有TGxxxA/GxG辅因子结合位点和YxxxK催化位点基序,含有SDR酶的活性位点和NAD(P)结合位点。通过体外酶促试验证实Dp3βHSD1/2均能催化孕烯醇酮生成洋地黄毒苷前体化合物黄体酮,且Dp3βHSD1较Dp3βHSD2具有更强的催化能力。基因表达水平分析表明Dp3βHSD1基因在叶中的表达水平显著高于Dp3βHSD2,而洋地黄毒苷在叶中特异性富集,基于以上结果推测洋地黄毒苷生物合成途径中孕烯醇酮脱氢生成黄体酮过程主要由Dp3βHSD1催化完成。该研究成功解析了功能性3β-羟基类固醇脱氢酶,为进一步解析洋地黄中强心苷类化合物的生物合成途径奠定基础。

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.