Mycobacterium Tuberculosis Isoprenoid Biosynthetic Pathway Ispd Ispe Enzymatic Function Study

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.