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Characterization of 4‐diphosphocytidyl‐2‐C‐methyl‐D‐erythritol synthase (IspD) from Mycobacterium tuberculosis

Many eubacteria, including Mycobacterium tuberculosis, utilize the 2‐C‐methyl‐D‐erythritol (MEP) pathway for the biosynthesis of isopentenyl diphosphate, a precursor of all isoprenoid compounds. This pathway is of interest as a source of potential drug targets since it is absent in mammals and disruption of the genes involved in Escherichia coli are lethal. In the MEP pathway, 4‐diphosphocytidyl‐2‐C‐methyl‐D‐erythritol is formed from MEP and cytidine 5′‐triphosphate (CTP), which is catalyzed by a 4‐diphosphocytidyl‐2‐C‐methyl‐D‐erythritol (CDP‐ME) synthase (IspD in E. coli). Blast searches show that the protein encoded by Rv3582c in M. tuberculosis has significant homology to E. coli IspD. Rv3582c was cloned and expressed and the purified protein is capable of catalyzing formation of CDP‐ME from MEP and CTP. The enzyme is active between pH 6.0 – 9.0 with optimal activity at pH 8.0 and requires divalent cations. Rv3582c has Km values of 54 μM and 49 μM for MEP and CTP, respectively. The Kcat and Kcat/Km values were calculated to be 3.29 × 104min−1 and 6.13 × 105M−1min−1 and 4.28 × 104 min−1 and 8.66 × 105M−1min−1for MEP and CTP, respectively. Allelic disruption of Rv3582c in M. tuberculosis provided direct evidence that the protein encoded by this gene is essential for M. tuberculosis survival. Thus, Rv3582c encodes a functional IspD in M. tuberculosis, and provides a new target for the development of novel anti‐tuberculosis drugs.