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Journal of Bacteriology, December 2005, p. 8395-8402, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8395-8402.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (IspC) from Mycobacterium tuberculosis: towards Understanding Mycobacterial Resistance to Fosmidomycin

Rakesh K. Dhiman,¹ Merrill L. Schaeffer,^1, Ann Marie Bailey,¹ Charles A. Testa,² Hataichanok Scherman,¹ and Dean C. Crick^1*

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523,¹ Echelon Biosciences, Inc., 675 Arapeen Drive, Suite 302, Salt Lake City, Utah 84108²

Received 23 May 2005/ Accepted 4 October 2005

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg²⁺ ions and exhibits optimal activity between pH 7.5 and 7.9; the K_m for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 µM, and the K_m for NADPH was 29.7 µM. The specificity constant of Rv2780c in the forward direction is 1.5 x 10⁶ M^–1 min^–1, and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.

Present address: Biological Research & Development, Boehringer Ingelheim Vetmedica, Inc., 2621 N. Belt Hwy., St. Joseph, MO 64506-2002.

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