Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase: potential for drug development

Abstract

Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 microM for MEP and 53.2 microM for CTP. Calculated kcat and kcat/Km values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.

FIG. 1.

MEP pathway. The reaction catalyzed by CDP-ME synthase (IspD) is highlighted. In M. tuberculosis, IPP and DMAPP are synthesized through the activities of a cascade of enzymes (DXS through IspH). DXS, 1-deoxy-d-xylulose 5-phosphate synthase; IspC, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase; IspE, 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; IspF, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; IspG, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IspH, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase. Identification numbers have been included for M. tuberculosis enzymes for which there is experimental evidence of activity.

FIG. 2.

Demonstration of ispD essentiality for M. tuberculosis H37Rv survival. Double-crossover strains were isolated in the wild-type and merodiploid backgrounds and analyzed using PCR primers IspDint1 and IspDint2 to amplify ispD. All strains in the wild-type background were wild type. Strains with either a wild-type or deletion allele were isolated in the merodiploid background. Lane M, lamdba HindIII marker; lanes 1 to 5, double-crossover strains generated from the merodiploid background; lane 6, wild-type genomic control; lane 7, delivery vector control. Lanes 1, 2, 4, and 5 show the product expected for a deletion strain, and lane 3 shows the product expected for a wild-type strain.

FIG. 3.

Purification of recombinant IspD. Analysis of protein fractions from E. coli transformed with pET28a(+)::Rv3582c is shown. Lane 1, cell lysates prior to IPTG treatment; lane 2, cell lysates after IPTG treatment; lane 3, purified His₆-IspD fraction after immobilized metal-affinity chromatography; lane 4, Western blot hybridization analysis of purified IspD using an anti-His antibody; lane M, molecular size markers (kDa). In lane 3, Rv3582c expression was visualized with Coomassie brilliant blue 250R.

FIG. 4.

Effects of pH and divalent cation concentration on M. tuberculosis IspD activity. (A) The optimal pH for catalytic activity was determined using MES (pH 5.5 to 6.5), MOPS (pH 6.5 to 7.5), Tris-HCl (pH 7.5 to 8.5), and TAPS (pH 8.5 to 9.5). (B) Divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, or Ca²⁺) were added to the reaction mixtures at the indicated concentrations. Reaction mixtures containing 38.5 pmol of purified IspD enzyme were prepared as described in Materials and Methods.