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

Abstract

ABSTRACT 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 Mg 2+ 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 × 10 6 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.