Cloning and Expression of Mycobacterium tuberculosis and Mycobacterium leprae Dihydropteroate Synthase in Escherichia coli

The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.

本研究通过从基因组DNA文库中扩增获得的探针进行杂交，成功分离得到结核分枝杆菌（Mycobacterium tuberculosis）与麻风分枝杆菌（Mycobacterium leprae）的二氢蝶酸合酶（dihydropteroate synthase）编码基因。DNA测序结果显示，结核分枝杆菌二氢蝶酸合酶的编码序列为一段长840 bp的开放阅读框，可编码含280个氨基酸的蛋白质；麻风分枝杆菌二氢蝶酸合酶的编码序列则为一段长852 bp的开放阅读框，可编码含284个氨基酸的蛋白质。上述两种二氢蝶酸合酶均在二氢蝶酸合酶缺陷型大肠杆菌（Escherichia coli）菌株中，于T5启动子的调控下完成表达。经三步色谱纯化流程后，结核分枝杆菌与麻风分枝杆菌来源的二氢蝶酸合酶均达到了98%以上的纯度。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测结果显示，结核分枝杆菌二氢蝶酸合酶的表观分子质量约为29 kDa，麻风分枝杆菌二氢蝶酸合酶的表观分子质量约为30 kDa。对两种酶进行凝胶过滤层析分析，结果显示其表观分子质量约为60 kDa，表明天然状态下的酶以由两个相同亚基组成的同源二聚体形式存在。本研究测定了结核分枝杆菌与麻风分枝杆菌二氢蝶酸合酶的稳态动力学参数。代表性磺酰胺类化合物及氨苯砜（dapsone）对这两种分枝杆菌来源的二氢蝶酸合酶均具有强效抑制活性；但作为潜在二氢蝶酸合酶抑制剂的抗分枝杆菌药物对氨基水杨酸（p-aminosalicylate），对该酶的抑制活性却较弱。