Lin Xiaohua, Xu Shengfeng, Yang Yanping, Wu Junchen, Wang Hongjun, Shen Hongbo, Wang Honghai
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PR China.
Protein Expr Purif. 2009 Mar;64(1):8-15. doi: 10.1016/j.pep.2008.09.020. Epub 2008 Oct 10.
The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis is the main reason why tuberculosis (TB) continues to be a major health problem worldwide. It is urgent to discover novel anti-mycobacterial agents based on new drug targets for the treatment of TB, especially MDR-TB. Tryptophan biosynthetic pathway, which is essential for the survival of M. tuberculosis and meanwhile absent in mammals, provides potential anti-TB drug targets. One of the promising drug targets in this pathway is anthranilate synthase component I (TrpE), whose role is to catalyze the conversion of chorismate to anthranilate using ammonia as amino source. In order to get a deep understanding of TrpE, a study on purification and characteristic identification of TrpE is required. In this work, the putative trpE gene of M. tuberculosis H37Rv was expressed as a fusion protein with a 6x His-tag on the N-terminal (His-TrpE) in Escherichia coli. The recombinant TrpE protein was successfully purified and then its enzymatic characteristics were analyzed. The native TrpE without His-tag was obtained by removal of the N-terminal fusion partner of His-TrpE using enterokinase. It was found that N-terminal fusion partner had little influence on TrpE catalytic activity. In addition, the key residues related to enzyme catalytic activity and that involved in l-tryptophan inhibition were predicted in the structure of M. tuberculosis H37Rv TrpE. These results would be beneficial to the designing of novel anti-TB drugs with high potency and selectivity.
结核分枝杆菌多重耐药（MDR）菌株的出现是结核病在全球范围内仍然是一个主要健康问题的主要原因。迫切需要基于新的药物靶点发现新型抗分枝杆菌药物来治疗结核病，尤其是耐多药结核病（MDR-TB）。色氨酸生物合成途径对结核分枝杆菌的生存至关重要，而在哺乳动物中不存在，这提供了潜在的抗结核药物靶点。该途径中一个有前景的药物靶点是邻氨基苯甲酸合酶组分I（TrpE），其作用是以氨作为氨基来源催化分支酸转化为邻氨基苯甲酸。为了深入了解TrpE，需要对TrpE进行纯化和特性鉴定研究。在这项工作中，结核分枝杆菌H37Rv的假定trpE基因在大肠杆菌中表达为N端带有6x His标签的融合蛋白（His-TrpE）。成功纯化了重组TrpE蛋白，然后分析了其酶学特性。使用肠激酶去除His-TrpE的N端融合伴侣，获得了无His标签的天然TrpE。发现N端融合伴侣对TrpE催化活性影响很小。此外，在结核分枝杆菌H37Rv TrpE的结构中预测了与酶催化活性相关以及参与L-色氨酸抑制的关键残基。这些结果将有助于设计高效且具有选择性的新型抗结核药物。
