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

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 M, and the K m for NADPH was 29.7 M. 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.

Structures of Mycobacterium tuberculosis 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase Provide New Insights into Catalysis

“…We have now also shown that the inhibitor fosmidomycin, at an enzyme concentration of 0.35 M and substrate concentrations of 0.2 mM DXP and 0.1 mM NADPH, inhibits the enzyme with an estimated IC 50 value of 80 nM. This is similar to values of 310 and 30 nM that have been published for the M. tuberculosis and E. coli enzymes, respectively (50,51). A double mutant of MtDXR, containing the substitutions D151N and E222Q (MtDXR NQ ), represents a completely inactive enzyme, emphasizing the importance of these residues in metal binding and catalysis.…”

“…The sequence corresponding to M. tuberculosis Rv2870c codes for a fully functional DXR (16,49,50). Our previous construct, which contained a C-terminal truncation of 20 amino acids, showed similar kinetic parameters to the fulllength and a proteolytically cleaved enzyme (49) truncated by 18 amino acids.…”

Design, Synthesis, and X-ray Crystallographic Studies of α-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase

“…11 A number of clinical studies have demonstrated that fosmidomycin in combination with clindamycin has efficacy and good tolerability in the treatment of P. falciparum malaria. [17][18][19] In 2005, Dhiman et al 20 showed that fosmidomycin inhibits M. tuberculosis DXR (MtDXR) with an IC 50 of 0.31 µM, but has no effect on M. tuberculosis cell growth. The antibacterial activity of fosmidomycin on E. coli has been shown to rely on a cAMP-dependent glycerol-3-phosphate transporter that allows uptake in that organism, 21 but which seems to be lacking in M. tuberculosis.…”

Metabolism of Plasma Membrane Lipids in Mycobacteria and Corynebacteria

“…Unlike most bacteria, mycobacteria contain multiple types of Pol-P. For example, M. smegmatis produces decaprenol phosphate (C50, Dec-P, [148]) and heptaprenol phosphate (C35, Hep-P, [149]). Polyprenols are thought to be synthesized via the condensation of two C5 lipids, isopentenyl diphosphate [150][151][152] and dimethylallyl diphosphate derived from the mevalonateindependent methylerythritol 4-phosphate (MEP) pathway. These reactions are catalyzed by prenol diphosphate synthases and two M. tuberculosis proteins, Rv2361c and Rv1086, acting sequentially, are thought to fulfill this role [148,149,[153][154][155].…”