2-c-methylerythritol-4-phosphate and fosmidomycin

Isoprenoids, a diverse group of compounds derived from the five-carbon building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), are essential for survival in all organisms. Animals synthesize their isoprenoids from mevalonic acid (MVA), whereas most pathogenic bacteria and the malaria parasites utilize a completely different pathway for IPP and DMAPP synthesis, the methylerythritol phosphate (MEP) pathway. Plants use both pathways for the synthesis of isoprenoid precursors. The recent elucidation of the MEP pathway has opened the possibility to develop new strategies against microbial pathogens. Novel immunotherapeutic agents can be developed based on the MEP pathway intermediates known to activate the proliferation of human V-delta-9 V-gamma-2 T-cells after infection by many pathogenic bacteria and protozoa. Moreover, the design of specific inhibitors of MEP pathway enzymes (which are highly conserved but show no homology to mammalian proteins) should result in herbicides and drugs with broad-spectrum antimicrobial activity without mechanism-based toxicity to humans. A good example is the cure of bacterial infections and malaria with fosmidomycin, a highly stable inhibitor of the MEP pathway. The use of plants as test systems has led to the identification of additional inhibitors such as ketoclomazone. Biochemical, genetic and crystallographic approaches with the MEP pathway enzymes are now starting to characterize the inhibition kinetics and identify which residues play a structural or catalytic role. Current efforts should eventually contribute to an effective drug designed to fight against microbial pathogens that show resistance to currently available agents.

Topics: Anti-Bacterial Agents; Antimalarials; Drug Design; Erythritol; Fosfomycin; Herbicides; Humans; Plants; Sugar Phosphates; Terpenes

Topics: Amino Acid Sequence; Arabidopsis; Bacteria; Computational Biology; Erythritol; Escherichia coli; Fosfomycin; Genomics; Indoles; Mevalonic Acid; Molecular Sequence Data; Plastids; Polyisoprenyl Phosphates; Sequence Homology, Amino Acid; Solanum lycopersicum; Sugar Phosphates

Nitric oxide (NO), a well-known signaling molecule plays an important role in abiotic and biotic stress-induced production of plant secondary metabolites. In this study, roles of NO in water stress-induced tanshinone production in Salvia miltiorrhiza hairy roots were investigated. The results showed that accumulations of four tanshinone compounds in S. miltiorrhiza hairy roots were significantly stimulated by sodium nitroprusside (SNP, a NO donor) at 100 μM. Effects of SNP were just partially arrested by the mevalonate (MVA) pathway inhibitor (mevinolin), but were completely inhibited by the 2-C-methyl-d-erythritol-4-phosphate pathway (MEP) inhibitor (fosmidomycin). The increase of tanshinone accumulation and the up-regulation of HMGR and DXR expression by PEG and ABA treatments were partially inhibited by an inhibitor of NO biosynthesis (Nω-nitro-L-arginine methyl ester (L-NAME)) and a NO scavenger (2-(4-Carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)). Simultaneously, NO generation in the hairy roots was triggered by PEG and ABA, and the effects were also arrested by c-PTIO and L-NAME. These results indicated that NO signaling probably plays a central role in water stress-induced tanshinone production in S. miltiorrhiza hairy roots. SNP mainly stimulated the MEP pathway to increase tanshinone accumulation.

Topics: Abietanes; Abscisic Acid; Dehydration; Droughts; Erythritol; Fosfomycin; Gene Expression Regulation, Plant; Lovastatin; Mevalonic Acid; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Plant Roots; Polyethylene Glycols; Salvia miltiorrhiza; Signal Transduction; Sugar Phosphates

S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism.

Topics: Alcohol Oxidoreductases; Biomass; Cell Death; Cellulase; Cyclohexane Monoterpenes; Dimethylallyltranstransferase; Down-Regulation; Erythritol; Fosfomycin; Mevalonic Acid; Models, Biological; Monoterpenes; Nicotiana; Plant Leaves; Protein Prenylation; Secondary Metabolism; Sesquiterpenes; Signal Transduction; Stress, Physiological; Sugar Phosphates

Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. Here we employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologues in plants and bacteria, other HAD proteins may be MEP pathway regulators.

Topics: Aldose-Ketose Isomerases; Antimalarials; Catalytic Domain; Cytoplasm; Drug Resistance; Erythritol; Fosfomycin; Genetic Complementation Test; Phosphoric Monoester Hydrolases; Plasmodium falciparum; Protein Conformation; Protozoan Proteins; Sugar Phosphates; Xylose

In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

Topics: Catharanthus; Chloroplasts; Cloning, Molecular; Cycloheximide; DNA, Complementary; Enzyme Inhibitors; Erythritol; Fosfomycin; Gene Expression; Gene Expression Regulation, Plant; Herbicides; Isoenzymes; Isoxazoles; Metabolic Networks and Pathways; Organ Specificity; Oxazolidinones; Paraquat; Plant Leaves; Plant Proteins; Plant Roots; Promoter Regions, Genetic; Proteolysis; Sugar Phosphates; Transferases

The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.

Topics: Androstadienes; Antimalarials; Biosynthetic Pathways; Cells, Cultured; Drug Resistance; Electron Transport; Erythritol; Erythrocytes; Fosfomycin; Humans; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Plasmodium falciparum; Protein Prenylation; Protein Transport; Protozoan Proteins; rab5 GTP-Binding Proteins; Schizonts; Sugar Phosphates; Terpenes; Transport Vesicles; Ubiquinone; Vacuoles; Wortmannin

Topics: Chryseobacterium; Drug Resistance, Multiple, Bacterial; Enterobacter cloacae; Erythritol; Escherichia coli; Fosfomycin; Klebsiella pneumoniae; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Sugar Phosphates; Terpenes

The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.

Topics: Bacillus subtilis; Butadienes; Carbon-Carbon Double Bond Isomerases; Cell Proliferation; Cytotoxins; Erythritol; Fosfomycin; Genetic Engineering; Hemiterpenes; Organophosphorus Compounds; Pentanes; Sequence Deletion; Sugar Phosphates; Terpenes

Fosmidomycin and its analogue FR-900098 are potent inhibitors of 1-deoxy-d-xylulose 5-phosphate reducto-isomerase (DXR), the second enzyme of the MEP pathway for the biosynthesis of isoprenoids. This paper describes the synthesis of analogues of the two reverse phosphonohydroxamic acids 3 and 4, in which the length of the carbon spacer is modified, the N-methyl group of 3 is replaced by an ethyl group, and the phosphate group is replaced by potential isosteric moieties, i.e., sulfonate or carboxylate functionalities. The potential of the synthesized analogues to inhibit the E. coli DXR was evaluated.

Topics: Aldose-Ketose Isomerases; Enzyme Inhibitors; Erythritol; Fosfomycin; Molecular Structure; Multienzyme Complexes; Organophosphonates; Oxidoreductases; Structure-Activity Relationship; Sugar Phosphates; Terpenes

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

Topics: Cells, Cultured; Cloning, Molecular; Erythritol; Fosfomycin; Gene Expression Regulation, Plant; Green Fluorescent Proteins; Molecular Sequence Data; Nicotiana; Oryza; Plant Proteins; Plants, Genetically Modified; Polyisoprenyl Phosphates; Protein Prenylation; Recombinant Fusion Proteins; Sugar Phosphates

D. salina is one of the recognized natural sources to produce beta-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of beta-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-beta-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 microM fosmidomycin, carotenogenesis and the synthesis of beta-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and beta-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.

Topics: beta Carotene; California; Carotenoids; Cells, Cultured; Chlorophyta; Erythritol; Fosfomycin; Lovastatin; Mexico; Pentosephosphates; Sugar Phosphates; Terpenes

In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.

Topics: Animals; Erythritol; Erythrocytes; Fosfomycin; Genes, Protozoan; Plasmodium falciparum; Polymerase Chain Reaction; Sugar Phosphates

Specific inhibitors of the MVA pathway (pravastatin) and the MEP pathway (fosmidomycin) were used to interfere with the biosynthetic flux which leads to the production of aphidicolin-like diterpene in leaf organ cultures of Scoparia dulcis. Treatment of leaf organs with fosmidomycin resulted in dose dependent inhibition of chlorophylls, carotenoids, scopadulcic acid B (SDB) and phytol production, and no effect on sterol production was observed. In response to the pravastatin treatment, a significant decrease in sterol and perturbation of SDB production was observed.

Topics: Anti-Bacterial Agents; Antioxidants; Aphidicolin; Carotenoids; Chlorophyll; Chromatography, Gas; Chromatography, High Pressure Liquid; Diterpenes; Erythritol; Fosfomycin; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Photosynthesis; Plant Leaves; Pravastatin; Scoparia; Seeds; Spectrophotometry, Ultraviolet; Sterols; Sugar Phosphates

It was investigated whether the methyl-erythritol phosphate (MEP) pathway that generates volatile isoprenoids and carotenoids also produces foliar abscisic acid (ABA) and controls stomatal opening. When the MEP pathway was blocked by fosmidomycin and volatile isoprenoid emission was largely suppressed, leaf ABA content decreased to about 50% and leaf stomatal conductance increased significantly. No effect of fosmidomycin was seen in leaves with constitutively high rates of stomatal conductance and in plant species with low foliar ABA concentration. In all other cases, isoprene emission was directly associated with foliar ABA, but ABA reduction upon MEP pathway inhibition was also observed in plant species that do not emit isoprenoids. Stomatal closure causing a midday depression of photosynthesis was also associated with a concurrent increase of isoprene emission and ABA content. It is suggested that the MEP pathway generates a labile pool of ABA that responds rapidly to environmental changes. This pool also regulates stomatal conductance, possibly when coping with frequent changes of water availability. MEP pathway inhibition by leaf darkening, and its down-regulation by exposure to elevated CO2, was also associated with a reduction of foliar ABA content. However, stomatal conductance was reduced, indicating that stomatal aperture is not regulated by the MEP-dependent foliar ABA pool, under these specific cases.

Topics: Abscisic Acid; Carbon Dioxide; Erythritol; Fosfomycin; Photosynthesis; Plant Leaves; Poaceae; Signal Transduction; Sugar Phosphates; Terpenes

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 microM, and the K(m) for NADPH was 29.7 microM. The specificity constant of Rv2780c in the forward direction is 1.5 x 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.

Topics: Aldose-Ketose Isomerases; Anti-Bacterial Agents; Coenzymes; Drug Resistance, Bacterial; Enzyme Inhibitors; Enzyme Stability; Erythritol; Fosfomycin; Genetic Complementation Test; Hydrogen-Ion Concentration; Magnesium; Multienzyme Complexes; Mycobacterium tuberculosis; NADP; Oxidoreductases; Pentosephosphates; Salmonella enterica; Sugar Phosphates

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In at least some plants (including Arabidopsis), common precursors are exchanged between the cytosol and the plastid. However, little is known about the signals that coordinate their biosynthesis and exchange. To identify such signals, we arrested seedling development by specifically blocking the MVA pathway with mevinolin (MEV) or the MEP pathway with fosmidomycin (FSM) and searched for MEV-resistant Arabidopsis mutants that also could survive in the presence of FSM. Here, we show that one such mutant, rim1, is a new phyB allele (phyB-m1). Although the MEV-resistant phenotype of mutant seedlings is caused by the upregulation of MVA synthesis, its resistance to FSM most likely is the result of an enhanced intake of MVA-derived isoprenoid precursors by the plastid. The analysis of other light-hyposensitive mutants showed that distinct light perception and signal transduction pathways regulate these two differential mechanisms for resistance, providing evidence for a coordinated regulation of the activity of the MVA pathway and the crosstalk between cell compartments for isoprenoid biosynthesis during the first stages of seedling development.

Topics: Alleles; Arabidopsis; Arabidopsis Proteins; Erythritol; Fosfomycin; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Hydroxymethylglutaryl CoA Reductases; Light; Lovastatin; Mevalonic Acid; Mutation; Photoreceptor Cells; Phytochrome; Phytochrome B; Signal Transduction; Sugar Phosphates; Terpenes; Transcription Factors

Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.

Topics: Animals; Antimalarials; Dolichols; Erythritol; Erythrocytes; Fosfomycin; Genes, Protozoan; Humans; Malaria, Falciparum; Molecular Structure; Pentosephosphates; Plasmodium falciparum; Pyridoxal Phosphate; Spectrometry, Mass, Electrospray Ionization; Sugar Phosphates; Ubiquinone

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

Topics: Cytosol; Erythritol; Fosfomycin; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mevalonic Acid; Nicotiana; Phytosterols; Plastids; Plastoquinone; Signal Transduction; Sugar Phosphates; Xylulose

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids. Recombinant E. coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: k(cat) = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM. The rearrangement/reduction is reversible; K(eq) = 45 +/- 6 for DXP and MEP at 150 microM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP.

Topics: Binding, Competitive; DNA Primers; Enzyme Inhibitors; Erythritol; Escherichia coli; Fosfomycin; Kinetics; NADP; Oxidation-Reduction; Pentosephosphates; Phenothiazines; Protein Binding; Recombinant Proteins; Substrate Specificity; Sugar Phosphates; Transferases

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.

Topics: Aldose-Ketose Isomerases; Animals; Carbon Radioisotopes; Carotenoids; Cells, Cultured; Cyperus; Cytochrome P-450 Enzyme Inhibitors; Electron Transport Complex IV; Enzyme Inhibitors; Erythritol; Fosfomycin; Insecta; Juvenile Hormones; Lovastatin; Mevalonic Acid; Miconazole; Models, Biological; Models, Chemical; Multienzyme Complexes; Oxidoreductases; Sesquiterpenes; Sugar Phosphates