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Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2-C-Methyl-D-Erythritol Synthase: Potential for Drug Development

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523,¹ Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts and the London NHS Trust, 4 Newark Street, London E1 2AT, United Kingdom,² Division of Biological Chemistry and Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom³

Received 12 June 2007/ Accepted 28 September 2007

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 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^–1 min^–1 for MEP and 1.0 min^–1 and 18.8 mM^–1 min^–1 for CTP, respectively.

Published ahead of print on 5 October 2007.