结核分支杆菌H_(37)Rv莽草酸脱氢酶基因的克隆、表达及酶学性质的研究

Cloning and Expression of Shikimate Dehydrogenase Gene from Mycobacterium tuberculosis H37Rv and Properties of the Recombinant Shikimate Dehydrogenase

以结核分支杆菌H3 7Rv基因组为模板 ,扩增了该菌株的莽草酸脱氢酶基因aroE ,通过在大肠杆菌BL2 1(DE3) pLYS中表达 ,纯化出可溶性的重组结核分支杆菌莽草酸脱氢酶 (SD) .对其酶学性质测定分析表明 :在辅酶NADPH作用下该酶可催化还原 3 脱氢莽草酸生成莽草酸 ,最适pH值为 11,最适温度为 6 3℃ ,比活性 8.2 6× 10 4U/mg .Mg2 + ,Ni2 + ,Zn2 + 等金属离子及NP4 0、TritonX 10 0等变性剂对其酶活有一定的促进作用 ,而SDS则强烈抑制该酶活性 .应用圆二色仪初步测定了重组蛋白的二级结构 ,重组SD的二级结构中大约有 2 9.2 %的α螺旋 ,9.3% β折叠 ,32 .7% β转角 ,2 8.8%无规卷曲 .结核分支杆菌H3 7Rv莽草酸脱氢酶基因的克隆 ,为该酶的晶体结构分析、免疫学研究及抗结核药物靶点的筛选奠定基础 .

The M.tuberculosis aroE -encoded shikimate dehydrogenase (SD) was cloned and expressed in Escherichia coli BL21(DE3)pLYS. The soluble and active M.tuberculosis recombinant SD has been obtained . SD catalyzes NADPH-dependent reduction of 3-dehydroshikimate to form shikimate and NADP^(+). The optimal pH, optimal temperature and enzyme activity of the recombinant protein were 11, 63 ℃ and 8.26×10~4 U/mg, respectively. The enzyme activity was promoted in the presence of Mg^(2+),Ni^(2+),Zn^(2+), Nonident P40 and TritonX-100 but inhibited substantially with SDS. Circular dichroism studies on the recombinant SD indicated that the secondary structure of the recombinant protein had about 29.2% α -Helix,9.3% β -Sheet,32.7% β -Turn,28.8% Random coil. The results have contributed to crystallization trials,immunologic reseach and screening of target sites of the M.tuberculosis SD.