A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.

We have solved the 2.5-Å crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-Derythritol-4-phosphate pathway of isoprenoid biosynthesis. The structure reveals that the enzyme is present as a homodimer. Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain. The connective domain is responsible for dimerization and harbors most of the active site. The strictly conserved acidic residues Asp 150 , Glu 152 , Glu 231 , and Glu 234 are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity. The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal. A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers. A possible involvement of this loop in an induced fit during substrate binding is discussed. Isoprenoids, formed by the condensation of varying numbers of isopentenyl diphosphate (IPP)1 units, constitute a major class of both primary and secondary metabolites including, for example, the ubiquitous sterols as well as dolichols, plastochinones, carotenoids, the prenyl side chains of chlorophylls, and ubiquinones (1). In archaea, fungi, and mammals, the central building block of isoprenoids, IPP, is formed from acetyl-CoA via the classical mevalonate pathway that was described in the 1950s (reviewed in Ref. 2). Only about 8 years ago, however, the existence of an alternative IPP biosynthesis pathway was established (3) (Fig. 1). The first step, in which the condensation of pyruvate and D-glyceraldehyde-3-phosphate leads to the formation of 1-deoxy-D-xylulose-5-phosphate (DOXP) under release of CO 2 , is catalyzed by DOXP synthase. Subsequently, DOXP reductoisomerase mediates an intramolecular rearrangement followed by reduction using NADPH as hydrogen donor with 2-C-methyl-D-erythritol-4-phosphate (MEP) as product. In addition to NADPH, this reaction depends on the presence of divalent cations, with Mn 2ϩ being most effective (4). In subsequent steps, MEP is cytidylated by the enzyme CDP-ME synthetase under release of pyrophosphate and phosphorylated by CDP-ME kinase to yield 4-diphosphocytidyl-2-Cmethyl-D-erythritol-2-phosphate (CDP-ME-2-phosphate). This intermediate is then cyclized by MECDP synthase under release of CMP, resulting in the formation of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MECDP) (reviewed in Ref. 5). The remaining terminal steps necessary for the synthesis of IPP are largely unknown as yet, although two genes controlling these steps, gcpE and lytB, have been characterized (6, 7). This second IPP biosynthesis pathway has been named the MEP pathway after its key meta...

The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB. ß

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.In all organisms studied so far, isoprenoids derive from the common isoprene units, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). In mammals and in fungi, IPP and DMAPP are formed exclusively by the mevalonate pathway (11). In contrast, many eubacteria (including Escherichia coli), algae, and the plastids of higher plants synthesize IPP and DMAPP by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (9, 34). The MEP pathway was also identified in a plastid-like organelle of malaria parasites (15). Since the MEP pathway is absent in humans, it has been validated as a drug target for the treatment of both bacterial and parasitic infections (15,29).The pathway initiates with the formation of 1-deoxy-D-xylulose 5-phosphate (DOXP) by condensation of pyruvate and D-glyceraldehyde 3-phosphate catalyzed by the DOXP synthase (Dxs) (1,6,20,22,24,25,35,38). DOXP is then converted by the DOXP reductoisomerase (Dxr) into MEP (Fig.

SUMMARYActivation of human Vc9/Vd2 T cells by many pathogens depends on the presence of small phosphorylated non-peptide compounds derived from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. We here demonstrate that in Escherichia coli mutants deficient in lytB, an essential gene of the MEP pathway, a potent Vc9/Vd2 T-cell activator accumulates by a factor of approximately 150 compared to wild-type E. coli. The compound responsible for the strong immunogenicity of this E. coli mutant was subsequently characterized and identified as a small pyrophosphorylated metabolite, with a molecular mass of 262 Da, derived from the MEP pathway. Stimulation of human peripheral blood mononuclear cells (PBMC) with extracts prepared from the lytB-deficient E. coli mutant led to upregulation of T-cell activation markers on the surface of Vc9/Vd2 T cells as well as proliferation and expansion of Vc9/Vd2 T cells. This response was dependent on costimulatory growth factors, such as interleukin (IL)-2, IL-15 and IL-21. Significant levels of interferon-c (IFN-c) and tumour necrosis factor-a (TNF-a) were secreted in the presence of IL-2 and IL-15, but not in the presence of IL-21, demonstrating that proliferating phosphoantigen-reactive Vc9/Vd2 T cells do not necessarily produce proinflammatory cytokines.

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The conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP) is effectively blocked by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) inhibitors such as the natural antibiotic fosmidomycin. Prediction of binding affinities for closely related Dxr ligands as well as estimation of the affinities of structurally more distinct inhibitors within this class of non-hydrolyzable phosphate mimics relies on the synthesis of fosmidomycin derivatives with a broad range of target affinity. Maintaining the phosphonic acid moiety, linear modifications of the lead structure were carried out in an effort to expand the SAR of this physicochemically challenging class of compounds. Synthetic access to a set of phosphonic acids with inhibitory activity (IC(50)) in the range from 1 to >30 microM vs. E. coli Dxr and 0.4 to 20 microM against P. falciparum Dxr is reported.

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