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 K-m values of 58.5 mu M for MEP and 53.2 mu M for CTP. Calculated k(cat) and k(cat)/K-m 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.
Original language | English |
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Pages (from-to) | 8922-8927 |
Number of pages | 6 |
Journal | Journal of Bacteriology |
Volume | 189 |
Issue number | 24 |
DOIs | |
Publication status | Published - Dec 2007 |
Keywords
- ESCHERICHIA-COLI
- ISOPRENOID BIOSYNTHESIS
- LETHAL MUTATIONS
- CELL-WALL
- GENE
- 1-DEOXY-D-XYLULOSE
- IDENTIFICATION
- 5-PHOSPHATE
- INTERMEDIATE
- MUTAGENESIS