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J. Bacteriol. doi:10.1128/JB.00925-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of the Mycobacterium tuberculosis 4–diphosphocytidyl–2–C–methyl–D–erythritol synthase: Potential for Drug Development

Mycobacterial Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, U.S.A., 80523; Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts and the London, 4 Newark Street, London E1 2AT, UK, Division of Biological Chemistry and Molecular Microbiology, College of Life Sciences,University of Dundee, Dundee DD1 5EH, Scotland, UK

^* To whom correspondence should be addressed. Email: Dean.Crick{at}colostate.edu.

	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 in humans and disruption of the responsible genes has shown a lethal phenotype in 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, which is catalyzed by a 4–diphosphocytidyl–2–C–methyl–D–erythritol synthase (IspD). In the present work we have demonstrated that Rv3582c is essential in M. tuberculosis, Rv3582c has been cloned, expressed, and the encoded protein purified. The purified M. tuberculosis IspD 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 9.0), with peak activity at pH 8.0. The activity is 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 conditions tested, M. tuberculosis IspD has K_m values of 58.5 µM for MEP and 53.2 µM for CTP. Calculated K_cat and K_cat/K_m values were 0.72 min^-1 and 12.3 mM^-1min^-1, for MEP and 1.0 min^-1 and 18.8 mM^-1min^-1 for CTP, respectively.