School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China.
School of Life science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China.
Int J Biol Macromol. 2018 Dec;120(Pt A):673-682. doi: 10.1016/j.ijbiomac.2018.08.165. Epub 2018 Aug 29.
Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3β-Hydroxysteroid dehydrogenase (3βHSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3βHSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3βHSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3βHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD-Bbg3βHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg-3βHSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg-3βHSD was highly homologous to the 3βHSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg-3βHSD and 3βHSD from Rana rugosa. The optimized expression of recombinant Bbg-3βHSD were achieved by inducing with 0.1 mmol L IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3β-oxidized by Bbg-3βHSD when NAD was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg-3βHSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L.
蟾毒甾烯类化合物是蟾蜍耳后腺分泌的主要活性成分之一。对中华大蟾蜍耳后腺组织高通量转录组测序的初步研究表明，3β-羟甾脱氢酶（3βHSD）在耳后腺中的表达量几乎是肝脏中的 20 倍。该酶 3βHSD 是甾体生成组织中孕激素、雌激素和雄激素等甾体生物合成的必需步骤。因此，3βHSD 可能是参与蟾毒甾烯类化合物生物合成的重要酶。本研究从中华大蟾蜍耳后腺克隆了 Bbg-3βHSD cDNA。利用基因工程技术构建了重组原核融合表达质粒 pCOLD-Bbg3βHSD，将其导入大肠杆菌 BL21 中进行原核表达。Bbg-3βHSD 含有 1134bp 的开放阅读框（ORF），编码 377 个氨基酸残基。推测蛋白分子量为 42.8kDa，理论等电点为 8.68。氨基酸序列同源性分析表明，Bbg-3βHSD 与其他物种的 3βHSD 蛋白高度同源。系统进化树显示，Bbg-3βHSD 与红腹锦蛙的 3βHSD 相似性最高。用 0.1mmol/L IPTG 在 15°C 诱导 20h 可实现重组 Bbg-3βHSD 的优化表达。体外酶活性表明，当 NAD 作为辅酶时，Bbg-3βHSD 可以将孕烯醇酮和脱氢表雄酮 3β-氧化。酶学性质表明，重组 Bbg-3βHSD 的最适反应温度为 40°C，最适 pH 值为 8.5，最适辅酶浓度为 1.5mmol/L。
