fosfomycin and isoprene

Isoprenoids are synthesized by consecutive condensations of their five-carbon precursor, isopentenyl diphosphate, to its isomer, dimethylallyl diphosphate. Two pathways for these precursors are known. One is the mevalonate pathway, which operates in eucaryotes, archaebacteria, and cytosols of higher plants. The other is a recently discovered pathway, the nonmevalonate pathway, which is used by many eubacteria, green algae, and chloroplasts of higher plants. To date, five reaction steps in this new pathway and their corresponding enzymes have been identified. EC numbers of these enzymes have been assigned by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) and are available at http://www.chem.qmw.ac.uk/iubmb/enzyme/reaction/terp/nonMVA.html.

Topics: Butadienes; Enzyme Inhibitors; Escherichia coli; Fosfomycin; Hemiterpenes; Mevalonic Acid; Pentanes; Polyisoprenyl Phosphates

Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

Topics: Alendronate; Biosynthetic Pathways; Butadienes; Diphosphonates; Elasticity; Fosfomycin; Hemiterpenes; Hybridization, Genetic; Kinetics; Light; Metabolic Flux Analysis; Pentanes; Photosynthesis; Photosystem II Protein Complex; Plant Leaves; Plastids; Populus; Substrate Specificity; Time Factors

The methylerythritol phosphate (MEP) pathway in plants produces the prenyl precursors for all plastidic isoprenoids, including carotenoids and quinones. The MEP pathway is also responsible for synthesis of approximately 600 Tg of isoprene per year, the largest non-methane hydrocarbon flux into the atmosphere. There have been few studies of the regulation of the MEP pathway in plants under physiological conditions. In this study, we combined gas exchange techniques and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) and measured the profile of MEP pathway metabolites under different conditions. We report that in the MEP pathway, metabolites immediately preceding steps requiring reducing power were in high concentration. Inhibition of the MEP pathway by fosmidomycin caused deoxyxylulose phosphate accumulation in leaves as expected. Evidence is presented that accumulation of MEP pathway intermediates, primarily methylerythritol cyclodiphosphate, is responsible for the post-illumination isoprene burst phenomenon. Pools of intermediate metabolites stayed at approximately the same level 10 min after light was turned off, but declined eventually under prolonged darkness. In contrast, a strong inhibition of the second-to-last step of the MEP pathway caused suppression of isoprene emission in pure N(2). Our study suggests that reducing equivalents may be a key regulator of the MEP pathway and therefore isoprene emission from leaves.

Topics: Acclimatization; Butadienes; Chromatography, High Pressure Liquid; Darkness; Erythritol; Fosfomycin; Hemiterpenes; Light; Mass Spectrometry; Metabolic Networks and Pathways; Metabolome; Nitrogen; Pentanes; Plant Extracts; Plant Leaves; Populus; Reference Standards; Time Factors

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.

Topics: Alkyl and Aryl Transferases; Biocatalysis; Blotting, Western; Butadienes; Carbon; Carbon Dioxide; Cell Extracts; Chloroplasts; Circadian Rhythm; Erythritol; Fosfomycin; Hemiterpenes; Isotope Labeling; Light; Metabolic Networks and Pathways; Models, Biological; Nicotiana; Pentanes; Photosynthesis; Plant Leaves; Plant Proteins; Plants, Genetically Modified; Temperature

After darkening, isoprene emission continues for 20 to 30 min following biphasic kinetics. The initial dark release of isoprene (postillumination emission), for 200 to 300 s, occurs mainly at the expense of its immediate substrate, dimethylallyldiphosphate (DMADP), but the origin and controls of the secondary burst of isoprene release (dark-induced emission) between approximately 300 and 1,500 s, are not entirely understood. We used a fast-response gas-exchange system to characterize the controls of dark-induced isoprene emission by light, temperature, and CO(2) and oxygen concentrations preceding leaf darkening and the effects of short light pulses and changing gas concentrations during dark-induced isoprene release in hybrid aspen (Populus tremula × Populus tremuloides). The effect of the 2-C-methyl-D-erythritol-4-phosphate pathway inhibitor fosmidomycin was also investigated. The integral of postillumination isoprene release was considered to constitute the DMADP pool size, while the integral of dark-induced emission was defined as the "dark" pool. Overall, the steady-state emission rate in light and the maximum dark-induced emission rate responded similarly to variations in preceding environmental drivers and atmospheric composition, increasing with increasing light, having maxima at approximately 40 °C and close to the CO(2) compensation point, and were suppressed by lack of oxygen. The DMADP and dark pool sizes were also similar through their environmental dependencies, except for high temperatures, where the dark pool significantly exceeded the DMADP pool. Isoprene release could be enhanced by short lightflecks early during dark-induced isoprene release, but not at later stages. Fosmidomycin strongly suppressed both the isoprene emission rates in light and in the dark, but the dark pool was only moderately affected. These results demonstrate a strong correspondence between the steady-state isoprene emission in light and the dark-induced emission and suggest that the dark pool reflects the total pool size of 2-C-methyl-d-erythritol-4-phosphate pathway metabolites upstream of DMADP. These metabolites are converted to isoprene as soon as ATP and NADPH become available, likely by dark activation of chloroplastic glycolysis and chlororespiration.

Topics: Butadienes; Carbon Dioxide; Darkness; Environment; Fosfomycin; Hemiterpenes; Kinetics; Models, Biological; Oxygen; Pentanes; Plant Leaves; Populus; Temperature; Time Factors

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

In higher plants, many isoprenoids are synthesised via the chloroplastic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Attempts to elucidate the function of individual isoprenoids have used the antibiotic/herbicidal compound fosmidomycin (3-[N-formyl-N-hydroxy amino] propyl phosphonic acid) to inhibit this pathway. Examination of the effect of fosmidomycin on the major components of photosynthesis in leaves of white poplar (Populus alba) and tobacco (Nicotiana tabacum) was made. Fosmidomycin reduced net photosynthesis in both species within 1 h of application, but only when photosynthesis was light-saturated. In P. alba, these reductions were confounded by high light and fosmidomycin inducing stomatal patchiness. In tobacco, this was caused by significant reductions in PSII chlorophyll fluorescence and reductions in V(cmax) and J(max). Our data indicate that the diminution of photosynthesis is likely a complex effect resulting from the inhibition of multiple MEP pathway products, resulting in photoinhibition and photo-damage. These effects should be accounted for in experimental design and analysis when using fosmidomycin to avoid misinterpretation of results as measured by gas exchange and chlorophyll fluorescence.

Topics: Butadienes; Carbon Dioxide; Chlorophyll; Fluorescence; Fosfomycin; Hemiterpenes; Herbicides; Nicotiana; Pentanes; Photosynthesis; Plant Leaves; Plant Stomata; Populus; Terpenes

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K(M)(DXP) = 104 microM, K(M)(NADPH) = 13 microM, k(cat)(DXP) = 2 s(-1), k(cat)(NADPH) = 1.3 s(-1)), an IC(50) for fosmidomycin of 247 nM, and a K(i) for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg(+2) as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.

Topics: Aldose-Ketose Isomerases; Anti-Infective Agents; Butadienes; Cations, Divalent; Cloning, Molecular; Fosfomycin; Francisella; Hemiterpenes; High-Throughput Screening Assays; Kinetics; Metabolic Networks and Pathways; Microbial Sensitivity Tests; Multienzyme Complexes; Oxidoreductases; Pentanes; Phosphorylation; Protein Structure, Tertiary; Recombinant Proteins; Structural Homology, Protein; Substrate Specificity

Isoprene and nitric oxide (NO) are two volatile molecules that are produced in leaves. Both compounds were suggested to have an important protective role against stresses. We tested, in two isoprene-emitting species, Populus nigra and Phragmites australis, whether: (1) NO emission outside leaves is measurable and is affected by oxidative stresses; and (2) isoprene and NO protect leaves against oxidative stresses, both singularly and in combination. The emission of NO was undetectable, and the compensation point was very low in control poplar leaves. Both emission and compensation point increased dramatically in stressed leaves. NO emission was inversely associated with stomatal conductance. More NO was emitted in leaves that were isoprene-inhibited, and more isoprene was emitted when NO was reduced by NO scavenger c-PTIO. Both isoprene and NO reduced oxidative damages. Isoprene-emitting leaves which were also fumigated with NO, or treated with NO donor, showed low damage to photosynthesis, a reduced accumulation of H(2)O(2) and a reduced membrane denaturation. We conclude that measurable amounts of NO are only produced and emitted by stressed leaves, that both isoprene and NO are effective antioxidant molecules and that an additional protection is achieved when both molecules are released.

Topics: Butadienes; Fosfomycin; Hemiterpenes; Nitric Oxide; Oxidative Stress; Ozone; Pentanes; Photosensitizing Agents; Plant Leaves; Plant Stomata; Poaceae; Populus; Volatile Organic Compounds

Isoprene reduces visible damage (necrosis) of leaves caused by exposure to ozone but the mechanism is not known. Here we show that in Phragmites leaves isoprene emission was stimulated after a 3-h exposure to high ozone levels. The photosynthetic apparatus of leaves in which isoprene emission was inhibited by fosmidomycin became more susceptible to damage by ozone than in isoprene-emitting leaves. Three days after ozone fumigation, the necrotic leaf area was significantly higher in isoprene-inhibited leaves than in isoprene-emitting leaves. Isoprene-inhibited leaves also accumulated high amounts of nitric oxide (NO), as detected by epifluorescence light microscopy. Our results confirm that oxidative stresses activate biosynthesis and emission of chloroplastic isoprenoid, bringing further evidence in support of an antioxidant role for these compounds. It is suggested that, in nature, the simultaneous quenching of NO and reactive oxygen species by isoprene may be a very effective mechanism to control dangerous compounds formed under abiotic stress conditions, while simultaneously attenuating the induction of the hypersensitive response leading to cellular damage and death.

Topics: Benzoates; Butadienes; Cell Death; Fosfomycin; Hemiterpenes; Imidazoles; Nitric Oxide; Nitroprusside; Oxidative Stress; Ozone; Pentanes; Plant Leaves; Poaceae

Isoprene emission from leaves is dynamically coupled to photosynthesis through the use of primary and recent photosynthate in the chloroplast. However, natural abundance carbon isotope composition (delta(13)C) measurements in myrtle (Myrtus communis), buckthorn (Rhamnus alaternus), and velvet bean (Mucuna pruriens) showed that only 72% to 91% of the variations in the delta(13)C values of fixed carbon were reflected in the delta(13)C values of concurrently emitted isoprene. The results indicated that 9% to 28% carbon was contributed from alternative, slow turnover, carbon source(s). This contribution increased when photosynthesis was inhibited by CO(2)-free air. The observed variations in the delta(13)C of isoprene under ambient and CO(2)-free air were consistent with contributions to isoprene synthesis in the chloroplast from pyruvate associated with cytosolic Glc metabolism. Irrespective of alternative carbon source(s), isoprene was depleted in (13)C relative to mean photosynthetically fixed carbon by 4 per thousand to 11 per thousand. Variable (13)C discrimination, its increase by partially inhibiting isoprene synthesis with fosmidomicin, and the associated accumulation of pyruvate suggested that the main isotopic discrimination step was the deoxyxylulose-5-phosphate synthase reaction.

Topics: Atmosphere; Butadienes; Carbon; Carbon Dioxide; Carbon Isotopes; Cytosol; Darkness; Fosfomycin; Glucose; Glycolysis; Hemiterpenes; Mucuna; Myrtus; Pentanes; Photosynthesis; Plant Leaves; Rhamnus

Isoprene (2-methyl-1,3-butadiene) is the most abundant biogenic hydrocarbon released from vegetation, and there is continuing interest in understanding its biosynthesis from photosynthetic precursors in leaf chloroplasts. We used on-line proton-transfer-reaction mass spectrometry (PTR-MS) to observe the kinetics of (13)C-labeling of isoprene following exposure to (13)CO(2) and then the loss of (13)C after a return to normal (12)CO(2) in oak ( Quercus agrifolia Nee) and cottonwood (Populus deltoides Barr.) leaves. Assignments of labeled isoprene species were verified by gas chromatography-mass spectrometry. For the first time, it was possible to observe the half-lives of individually (13)C-labeled isoprene species during these transitions, and to trace some of the label to a C3 fragment that contained the two isoprene carbons derived from pyruvate via the deoxyxylulose-5-phosphate (DOXP) pathway. At steady state (under (13)CO(2)), approximately 80% of isoprene carbon was labeled, with fully labeled isoprene as the major species (approx. 60%). The source of the unlabeled C is suggested to be extrachloroplastic, but not from photorespiratory carbon. After a transfer to (12)CO(2), (13)C-labeling persisted in one isoprene carbon for several hours; this persistence was much more pronounced in (i) leaves inhibited by fosmidomycin, a specific inhibitor of the DOXP pathway, and (ii) in sun leaves which have higher ratios of soluble sugars to starch. From the mass 41-44 fragment data, and labeling predicted from the DOXP pathway in chloroplasts, precursors may arise from cytosolic pyruvate/phospho enolpyruvate equivalents transported into the chloroplast; this idea was supported by an indirect measure of pyruvate labeling. Other sources of cytosolic isoprene precursors (i.e. dimethylallyl diphosphate or pentose phosphate) could not be excluded. The data obtained shed light on the half-lives of photosynthetic metabolites, exchanges of carbon between cellular pools, and suggest multiple origins of isoprene precursors in leaves.

Topics: Algorithms; Butadienes; Carbon Dioxide; Carbon Isotopes; Chloroplasts; Cytosol; Fosfomycin; Gas Chromatography-Mass Spectrometry; Half-Life; Hemiterpenes; Light; Lovastatin; Models, Biological; Oxygen; Oxygen Consumption; Pentanes; Photosynthesis; Plant Leaves; Populus; Pyruvic Acid; Quercus

Isoprene is synthesized and emitted in large amounts by a number of plant species, especially oak (Quercus sp.) and aspen (Populus sp.) trees. It has been suggested that isoprene improves thermotolerance by helping photosynthesis cope with high temperature. However, the evidence for the thermotolerance hypothesis is indirect and one of three methods used to support this hypothesis has recently been called into question. More direct evidence required new methods of controlling endogenous isoprene. An inhibitor of the deoxyxylulose 5-phosphate pathway, the alternative pathway to the mevalonic acid pathway and the pathway by which isoprene is made, is now available. Fosmidomycin eliminates isoprene emission without affecting photosynthesis for several hours after feeding to detached leaves. Photosynthesis of fosmidomycin-fed leaves recovered less following a 2-min high-temperature treatment at 46 degrees C than did photosynthesis of leaves fed water or fosmidomycin-fed leaves in air supplemented with isoprene. Photosynthesis of Phaseolus vulgaris leaves, which do not make isoprene, exhibited increased thermotolerance when isoprene was supplied in the airstream flowing over the leaf. Other short-chain alkenes also improved thermotolerance, whereas alkanes reduced thermotolerance. It is concluded that thermotolerance of photosynthesis is a substantial benefit to plants that make isoprene and that this benefit explains why plants make isoprene. The effect may be a general hydrocarbon effect and related to the double bonds in the isoprene molecule.

Topics: Acclimatization; Butadienes; Fosfomycin; Hemiterpenes; Hot Temperature; Magnoliopsida; Pentanes; Photosynthesis; Plant Leaves; Temperature; Trees

Many plants invest carbon to form isoprene. The role of isoprene in plants is unclear, but many experiments showed that isoprene may have a role in protecting plants from thermal damage. A more general antioxidant action has been recently hypothesized on the basis of the protection offered by exogenous isoprene in nonemitting plants exposed to acute ozone doses. We inhibited the synthesis of endogenous isoprene by feeding fosmidomycin and observed that Phragmites australis leaves became more sensitive to ozone than those leaves forming isoprene. Photosynthesis, stomatal conductance, and fluorescence parameters were significantly affected by ozone only in leaves on which isoprene was not formed. The protective effect of isoprene was more evident when the leaves were exposed for a long time (8 h) to relatively low (100 nL L(-1)) ozone levels than when the exposure was short and acute (3 h at 300 nL L(-1)). Isoprene quenched the amount of H(2)O(2) formed in leaves and reduced lipid peroxidation of cellular membranes caused by ozone. These results indicate that isoprene may exert its protective action at the membrane level, although a similar effect could be obtained if isoprene reacted with ozone before forming active oxygen species. Irrespective of the mechanism, our results suggest that endogenous isoprene has an important antioxidant role in plants.

Topics: Antioxidants; Butadienes; Cell Membrane; Fosfomycin; Hemiterpenes; Hydrogen Peroxide; Lipid Peroxidation; Oxidative Stress; Ozone; Pentanes; Photosynthesis; Plant Leaves; Poaceae; Reactive Oxygen Species