【作者】 郑丽娜；

【导师】 张后今；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2011， 硕士

【摘要】 作为在亚洲广泛使用的农用抗生素和多种医用药物的前体,井冈霉素在农业及医学上具有非常重要的应用价值。因此,近年来井冈霉素的开发和研究是工业界和学术界倍受关注的问题。工业上发酵条件的优化,使得其每升产量增加了9倍,大大降低了井冈霉素的价格。同时,井冈霉素生物合成途径的研究证明了合成所必需的关键酶的存在,但是这些酶的结构和催化机理至今尚不明确。本课题首先运用基因克隆技术将GST片段及TEV酶切位点片段重组入pET-28a(+)载体以构建新型原核表达载体pET-28a-GST-TEV,再将目的片段valL克隆到上述载体并于大肠杆菌BL21(DE3) Rosetta中进行蛋白表达,随后将表达的蛋白用于结晶,并解析晶体结构。按照上述技术路线,本课题成功得到了带有His标签和GST标签的新型原核表达载体pET-28a-GST-TEV,大大提高了真核蛋白异源表达的可溶性,并能有效帮助蛋白折叠,具有较高的推广使用价值。同时本课题获得了高质量的ValL及其硒代衍生物晶体结构数据,其中衍射分辨率分别为2.0?和1.7?,成功解析出了ValL及ValL/海藻糖复合物的结构模型。结果表明ValL的整体结构与经典的“GT-B”葡萄糖转移酶非常相似,它含有两个结构域,每个结构域含有一个β/α/βRossman折叠,其活性中心位于两结构域的交界处。同时发现在ValL结构C端区域的最后两个螺旋间有一个拐点,使得螺旋上的氨基酸残基与N端区域相互作用,这也是GT-B酶家族的共同特点。此外,N端和C端交界处的电子云密度图很清楚地显示了ValL产物类似物海藻糖的位置。本课题阐明了井冈霉素生物合成8个必需基因之一valL编码的7-磷酸有效氧胺合成酶的结构,为进一步了解井冈霉素的合成机理奠定了基础,同时也为在农业、医学等相关方面深度利用井冈霉素提供了理论切入点。更多还原

【Abstract】 As a widely-used agricultural antibiotic in Asia and a precursor of various medicines, validamycin A bears extraordinary value for agriculture as well as pharmaceutical industry. Therefore, in recent years, the research and development of validamycin A has drawn significant attention from academia and industry alike. The optimization of fermentation condition has lead to ten-fold increase in yield per liter, which caused large drop in the price of validamycin A. Meanwhile, the study on its biosynthesis pathway has demonstrated the essential enzymes required for the validamycin A production. However, the structures and mechanisms of these enzymes on the pathway remain unclear. In this study, in order to construct the new prokaryotic expression vector pET-28a-GST-TEV we cloned the gene of GST and the cleavage site of TEV protease into pET-28a(+) plasmid. Then the target gene valL was inserted into pET 28a-GST-TEV and the recombinant protein was expressed in BL21(DE3) Rosetta cells. Subsequently, the protein was used to grow crystals and x-rays diffraction was chosen as the method to determine the three-dimensional structure.Using the protocol described above, we construct a novel prokaryotic expression vector pET-28a-GST-TEV featuring His tag and GST Tag. It would have good application in the hetero expression of eukaryotic genes. In addition, we collected the diffraction data of native ValL crystals as well as selenomethionine-labelled ValL crystals with resolutions of 1.9? and 1.7? respectively. Based on the above data, we built the three dimensional models of ValL and ValL/trehalose complex. The structural analysis shows that the overall structure of ValL closely resembles the classic fold of‘‘GT-B’’glycosyltransferases. It harbors two domains each of which contains aβ/α/βRossmann fold and an active site at the interface of them. Also found in the ValL structure is a kink between last two C-terminal helices, which is the common feature of GT-B enzyme family. The kink directs the last C-terminal helix into the vicinity of N-terminal domain and causes the residues in the helix to interact with the N-terminal domain. Furthermore, the electron density at the junction of N-terminal and C-terminal unambiguously indicates the location of trehalose embedded in the active site.In this work, we solved the structure of validoxylamine 7-phosphate synthase encoded by one of the eight essential genes required for validamycin biosynthesis. The results laid the framework for the further study of synthetic mechanism of validamycin and they are instrumental for the advanced application of validamycin in agriculture as well as medical science.更多还原