Biosynthesis of Isoprenoids: Characterization of a Functionally Active Recombinant 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv

State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China.
Journal of biochemistry and molecular biology (Impact Factor: 2.02). 12/2007; 40(6):911-20. DOI: 10.5483/BMBRep.2007.40.6.911
Source: PubMed


Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.

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    • "In M. tuberculosis, the MEP pathway begins with the condensation of pyruvic acid and glyceraldehyde-3-phosphate (Eoh et al., 2007), resulting in the final products IPP and its isomer, DMAPP (Eoh et al., 2007; Illarionova et al., 2006; Richard et al., 2004; Shi et al., 2007; Testa et al., 2006). This pathway is catalyzed by the eight enzymes 1- deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5- phosphate reductoisomerase (IspC), 4-diphosphocytidyl-2-C-methyl- D-erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl-D-ery- thritol-2-phosphate synthase (IspE), 2-C-methyl-D-erythritol 2,4- cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), 1-hydroxy-2-methyl-2-(E)-butenyl-4- diphosphate reductase (IspH), and isopentenyl diphosphate isomerase (Idi) (Eoh et al., 2009, 2007). "
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    ABSTRACT: Tuberculosis is a serious threat to world-wide public health usually caused in humans by Mycobacterium tuberculosis (M. tuberculosis). It exclusively utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the precursors of all isoprenoid compounds. The 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD; EC is the key enzyme of the MEP pathway. It is also of interest as a new chemotherapeutic target, as the enzyme is absent in mammals and ispD is an essential gene for growth. A high-throughput screening method was therefore developed to identify compounds that inhibit IspD. This process was applied to identify a lead compound, domiphen bromide (DMB), that may effectively inhibit IspD. The inhibitory action of DMB was confirmed by over-expressing or down-regulating IspD in Mycobacterium smegmatis (M. smegmatis), demonstrating that DMB inhibit M. smegmatis growth additionally through an IspD-independent pathway. This also led to higher levels of growth inhibition when combined with IspD knockdown. This novel IspD inhibitor was also reported to exhibit antimycobacterial activity in vitro, an effect that likely occurs as a result of perturbation of cell wall biosynthesis.
    European journal of pharmacology 09/2012; 694(1-3):45-52. DOI:10.1016/j.ejphar.2012.08.012 · 2.53 Impact Factor
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    ABSTRACT: Early and accurate detection of drug resistant Mycobacterium tuberculosis can improve both the treatment outcome and public health control of tuberculosis. A number of molecular-based techniques have been developed including ones using probe molecules that target drug resistance-related mutations. Although these techniques are highly specific and sensitive, mixed signals can be obtained when the drug resistant isolates are mixed with drug susceptible isolates. In order to overcome this problem, we developed a new drug susceptibility test (DST) for one of the most effective anti-tuberculosis drug, isoniazid. This technique employed a microplate hybridization assay that quantified signals from each probe molecule, and was evaluated using clinical isolates. The evaluation analysis clearly showed that the microplate hybridization assay was an accurate and rapid method that overcame the limitations of DST based on conventional molecular techniques.
    BMB reports 03/2009; 42(2):81-5. DOI:10.5483/BMBRep.2009.42.2.081 · 2.60 Impact Factor
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    ABSTRACT: Isoprenoids produced by bacteria are of particular interest as they encompass huge structural and functional diversity. A rapidly increasing number of bacterial derived isoprenoids have been reported in recent years, as many of the genes and biosynthetic gene clusters are responsible for their biosynthesis. Polyprenyl chains, synthesized by the condensation of isopentenyl diphosphate units, serve as the substrates for cyclases and subsequent tailoring enzymes. It is these enzymes, particularly the cyclases, which are responsible for the structural diversity of this chemical class. Recent studies have revealed novel insights into isoprenoid biosynthesis, and in several cases enzymatic mechanisms have been redefined.
    Current opinion in chemical biology 04/2009; 13(2):180-8. DOI:10.1016/j.cbpa.2009.02.029 · 6.81 Impact Factor
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