Herl Vanessa, Frankenstein Jördis, Meitinger Nadine, Müller-Uri Frieder, Kreis Wolfgang
Lehrstuhl für Pharmazeutische Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Planta Med. 2007 Jun;73(7):704-10. doi: 10.1055/s-2007-981537. Epub 2007 Jun 12.
During the biosynthesis of cardiac glycosides, Delta (5)-3beta-hydroxysteroid dehydrogenase (3 beta HSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3beta-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3 beta HSD gene was isolated from leaves of Digitalis lanata. It consisted of 870 nucleotides containing a 90 nucleotide long intron. A full-length cDNA clone that encodes 3 beta HSD was isolated by RT-PCR from the same source. A SPH I /KPN I 3 beta HSD cDNA was cloned into the pQE30 vector and then transferred into E. COLI strain M15[pREP4]. 3 beta HSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3 beta HSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). pQ3 beta HSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3beta-hydroxypregnanes but not cholesterol were 3beta-oxidised by pQ3 beta HSD when NAD was used as the co-substrate. Testosterone (4-androsten-17beta-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3 beta HSD has also 17beta-dehydrogenase activity. pQ3 beta HSD was able to reduce 3-keto steroids to their corresponding 3beta-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3 beta HSD genes were isolated and sequenced from another 6 species of the genus DIGITALIS. Gene structure and the deduced 3 beta HSD proteins share a high degree of similarity.
在强心苷的生物合成过程中，Δ(5)-3β-羟基类固醇脱氢酶（3βHSD，EC 1.1.1.51）将孕烯醇酮（5-孕烯-3β-醇-20-酮）转化为异孕酮（5-孕烯-3,20-二酮）。从毛花洋地黄的叶子中分离出一个3βHSD基因。它由870个核苷酸组成，包含一个90个核苷酸长的内含子。通过RT-PCR从同一来源分离出一个编码3βHSD的全长cDNA克隆。将一个SPH I /KPN I 3βHSD cDNA克隆到pQE30载体中，然后转移到大肠杆菌菌株M15[pREP4]中。3βHSD cDNA作为由273个氨基酸组成的His标签融合蛋白（pQ3βHSD）（计算分子量为28,561 Da）进行功能表达。pQ3βHSD通过在Ni-NTA上的金属螯合亲和层析进行纯化。当使用NAD作为共底物时，孕烯醇酮和其他3β-羟基孕烷但不是胆固醇被pQ3βHSD进行3β-氧化。睾酮（4-雄烯-17β-醇-3-酮）被转化为4-雄烯-3,17-二酮，表明pQ3βHSD也具有17β-脱氢酶活性。当使用NADH作为共底物时，pQ3βHSD能够将3-酮类固醇还原为其相应的3β-羟基衍生物。为了进行比较，从洋地黄属的另外6个物种中分离并测序了3βHSD基因。基因结构和推导的3βHSD蛋白具有高度相似性。
