结核分枝杆菌类异戊二烯合成途径中IspD和IspE酶学功能研究

【作者】 施文钧；

【导师】 王洪海；

【作者基本信息】 复旦大学 ， 分子免疫学， 2007， 博士

【摘要】 结核病是由结核分枝杆菌引起的一种严重的人类传染性疾病。目前全球人口中大约1/3的人已感染结核分枝杆菌,每年约有300万人死于结核。近年来,耐药结核杆菌的出现,使寻找新型抗结核杆菌的药物势在必行。2-C-甲基-D-赤藓糖醇-4-磷酸（MEP）途径用于合成异戊酰焦磷酸（IPP）或其异构物,二甲烯丙基焦磷酸（DMAPP）,这两者是胞内重要化合物类异戊二烯合成的共同前体。MEP途径是细菌生长所必需而人类不存在的代谢途径,因此成为筛选新型抗菌药物的潜在靶标。本文对结核分枝杆菌基因组中编码MEP途径的第三个酶,4-二磷酸胞苷2-C-甲基-D赤藓糖醇（CDP-ME）合成酶基因（ispD/Rv3582c）进行克隆,并表达纯化了该蛋白（MtIspD）。通过HPLC和非变性胶电泳分析,表明具生物活性的MtIspD蛋白为同二聚体。酶活分析显示MtIspD是金属酶,Mg²⁺为最适二价金属离子。MtIspD对辅因子底物特异性高,CTP为最适底物。CTP和MEP的Km值分别为92μM和43μM,Vmax为7.8μmol.min^-1.mg^-1,酶单体的转换数为3.4 S^-1。MtIspD酶热稳定性差,通过圆二色仪分析不同温度下的二级结构和三级结构,显示三维构象随温度的变化与酶活的丧失密切相关。对MtIspD模建结构与大肠杆菌结晶结构比较分析,显示MtIspD酶活性位点保守,酶单体β臂的相互作用对二聚体形成和酶活性中心形成起关键作用。进一步对酶活中心的三个保守碱性氨基酸和86位苏氨酸定点突变分析,结果显示K27A,R157A和K215A不仅使酶失活,而且影响蛋白二级结构和三维构象;86位苏氨酸定点突变（T86S和T86A）说明86位氨基酸羟基对MtIspD酶活有重要作用。我们对MtIspD酶活和结构的研究表明IspD蛋白家族比较保守,可以以IspD酶活中心筛选和设计广谱抗菌素,并且针对同时参与活性位点和二聚体形成的保守碱基似乎对抑制酶活更为有效。我们的研究为以该酶作为抗结核杆菌药物靶标筛选抑制剂和新型药物奠定了基础。结核分枝杆菌基因组中ispE基因（Rv1011）编码MEP途径的第四个酶4-二磷酸胞苷2-C-甲基-D赤藓糖醇（CDP-ME）激酶,由于ispE基因单独分布,但编码蛋白必须与催化上下游步骤的单功能IspD/IspF或双功能蛋白IspDF相互作用,才能使MEP途径进行,所以在MEP途径中起关键作用,因而是重要的抗结核杆菌药物靶标。然而MEP途径作为候选药物靶标,目前仍无亚细胞定位的实验报道。本文将ispE基因克隆到pET28a载体,在大肠杆菌BL21（DE3）中表达重组蛋白（MtIspE）,用Ni-NTA柱亲和层析纯化重组蛋白。其纯度和分子量分别通过SDS-PAGE和高效液相-质谱测定。利用圆二色仪对MtIspE进行二级结构测定,该重组蛋白中含有的α螺旋,β折叠,β转角和无规则卷曲分别为36.3%,26.9%,11.8%和25.1%。MtIspE酶活分析首先需制备CDP-ME底物,由上游重组蛋白MtIspD催化CTP和MEP形成,经高效液相/质谱联用纯化和分析产物,证实获得有效的CDP-ME。酶活测定双底物ATP和CDP-ME的Km值,分别为213μM and 317μM,Vmax为0.413μmol.min^-1.mg^-1,酶单体的转换数为0.23 S^-1。我们用MtIspE免疫大白兔获得高滴度的多克隆抗体,通过RT-PCR和蛋白印迹分析,说明结核分枝杆菌对数生长期确实表达IspE蛋白,该蛋白主要在结核分枝杆菌胞浆发挥最用。通过对MtIspE酶学分析和在结核分枝杆菌亚细胞定位的研究,将为筛选和修饰抗MtIspE的新型抗结核药物有一定的帮助。更多还原

【Abstract】 Tuberculosis,caused by Mycobacterium tuberculosis,is one of the most effective bacterial human pathogen.Over one-third of the world’ s population is infected with M.tuberculosis and almost 3 million people die from tuberculosis annually.Compounding the problem,strains of M. tuberculosis that are resistant to the major drugs used to treat tuberculosis are rapidly emerging worldwide.The eradication of tuberculosis requires the development of novel anti-mycobacterial agents for therapeutic treatment of M.tuberculosis.Although isoprenoids have enormous structural and functional complexities in nature,they have common precursors;isopentenyl diphosphate（IPP）or its isomer dimethylallyl diphosphate（DMAPP）.IPP and DMAPP are synthesized from the 2-C-methyl-D-erythritol 4-phosphate （MEP）pathway in bacteria.Given the essential nature of the MEP pathway in bacteria and the absence of this pathway in mammals,the enzymes comprising the MEP pathway represent potential targets for the generation of selective anti-mycobacterial molecules.4-diphosphocytidyl-2-C-methylerythritol synthetase（IspD）catalyzes the third step in the MEP pathway and is an attractive target for antibacterial drug design.Based on our general interest for the development of anti-tuberculosis agents,we first expressed,purified and characterized IspD from M.tuberculosis H37Rv（MtIspD）.Native MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa.The enzyme has high specificity for pyrimidine bases and narrow divalent cation requirements in its activity,with the maximal activity found in the presence of CTP and Mg²⁺.The Km values for MEP and CTP are 43μM and 92μM,respectively.In addition,MtIspD homology model was built using Thermotoga maritime IspD as a template.Compared with EcIspD,MtIspD molecular model shares conservation of the catalytic core with EcIspD, but displays flexibility at dimer interface.On the other hand,we identified that MtIspD is thermosensitive.Its tertiary conformational alteration is responsible for sharp loss of enzymatic activity.Because basic residues are dominating in the active core of IspD family,Lys27 （K27A）,Arg157（R157A）,Arg215（K215A）coupled with Thr86（T86A and T86S） were constructed by site-directed mutagenesis.K27A,R157A and K215A completely abolished enzyme activities,and influence the secondary structure and the tertiary structure.T86A decreased activity by 98%and T86S increased activity slightly（105%）,suggesting the hydroxyl group of Thr86 was required for catalytic process.This well characterized MtIspD would provide useful information in the screening of broad -spectrum hibitors using MtIspD as a target.The inhibitors targeting the conservative residues involved in formation of both active site and dimer of MtIspD may be more effective.The gene of 4-diphosphocytidyl-2-C-methyl-D-erythritol（CDP-ME） kinase encodes the forth enzyme of the MEP pathway.The gene of 4-diphosphocytidyl-2-C-methyl-D-erythritol（CDP-ME）kinase is separated from other genes of the MEP pathway in M.tuberculosis,but interacted closely with monofunctional protein IspD/IspF or bifunctioal protein MtlspDF.In addition,despite considerable progress in the characterization of MEP pathway,there is little information on the nature of the proteins associated with the subcelluar fraction.In the study, CDP-ME kinase gene from M.tuberculosis（ispE/Rv1011）was expressed in E.coli BL21（DE3）strain.The protein was purified to homogeneity by the Ni-NTA affinity chromatography.The purity and molecular weight were determined by SDS-PAGE and HPLC/MS.Its correct folding was verified by circular dichroism（CD）spectroscopy.The percentages forα-helix,β-sheet,β-turn,and random coil were 36.3%,26.9%,11.8%,and 25.1%, respectively.The enzyme assays revealed that the Km for CDP-ME and ATP were and 213μM and 317μM,respectively.Vmax was 0.413μmol.min^-1.mg^-1, the turnover number of subunit was 0.23 S^-1.Recombinant protein MtIspE was used for antiserum production.All subcellular fractions from M. tuberculosis were detected by multi-clonal MtIspE antibody.Subcellular fractionation of M.tuberculosis located the IspE protein in the cytoplasmic.This study may provide some information for the selection and modification of the inhibitors against MtIspE.更多还原