Qin Ning, Shen Yanbing, Yang Xu, Su Liqiu, Tang Rui, Li Wei, Wang Min
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
World J Microbiol Biotechnol. 2017 Jul;33(7):146. doi: 10.1007/s11274-017-2310-x. Epub 2017 Jun 20.
3-Ketosteroid-Δ-dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.
新金色分枝杆菌的3-酮甾体-Δ-脱氢酶（KsdD）可将雄甾-4-烯-3,17-二酮（AD）转化为雄甾-1,4-二烯-3,17-二酮。该反应对药用甾体产物有重大影响。新金色分枝杆菌KsdD的晶体结构和活性位点残基信息尚未可知，这导致对KsdD的工程改造很繁琐。在本研究中，通过蛋白质建模和定点诱变的方法，我们发现，来自FAD结合结构域的Y122、Y125、S138、E140和Y541以及来自催化结构域的Y365在该转化过程中起关键作用。与野生型相比，突变体AD转化率的下降表明Y125、Y365和Y541对KsdD的功能至关重要。Y122、S138和E140有助于KsdD的催化作用。以下分析揭示了新金色分枝杆菌KsdD中这些突变的催化机制。此处提供的这些信息有助于操控该酶的催化特性，以改善其在药用甾体工业中的应用。
