Shi Wenjun, Feng Jianfang, Zhang Min, Lai Xuhui, Xu Shengfeng, Zhang Xuelian, Wang Honghai
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China.
J Biochem Mol Biol. 2007 Nov 30;40(6):911-20. doi: 10.5483/bmbrep.2007.40.6.911.
Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.
由结核分枝杆菌引起的结核病仍然是人类主要的传染病之一。发现新的药物靶点以开发抗结核药物迫在眉睫。类异戊二烯生物合成的2-C-甲基-D-赤藓糖醇-4-磷酸（MEP）途径因其在细菌中的必需性和在哺乳动物中的缺失，被认为是发现新型抗生素的有吸引力的靶点。MEP胞苷转移酶（IspD）是该途径的第三步酶，催化MEP和CTP形成4-二磷酸胞苷-2-C-甲基赤藓糖醇（CDP-ME）和焦磷酸。在这项工作中，克隆并表达了结核分枝杆菌H37Rv的ispD基因（MtIspD）。通过N端融合组氨酸标签序列，MtIspD可通过一步镍亲和层析纯化至均一性。MtIspD以同源二聚体形式存在，表观分子量为52 kDa。酶学性质分析表明，MtIspD对嘧啶碱基具有高特异性，对二价阳离子的需求较窄，在CTP和Mg(2+)存在下活性最高。MtIspD的周转数为3.4 s(-1)。MEP和CTP的Km分别为43和92 μM。此外，MtIspD在50℃以上表现出热不稳定性。圆二色光谱表明，三级构象的改变与较高温度下酶活性的急剧丧失密切相关。本研究有望有助于更好地了解IspD的特性，并为开发治疗结核分枝杆菌的新型抗生素提供有用信息。
