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

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the meval-onate-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, includingMycobacterium 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 character-ized. Rv2870c from M. tuberculosis converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phos-phate 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 Km for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 M, and the Km for NADPH was 29.7 M. The specificity constant of Rv2780c in the forward direction is 1.5 106 M1 min1, 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. Fosmidomycin is a phosphonate antibiotic that is reported t