Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.

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.

Application of modified Lineweaver-Burk plots to studies of kinetics and regulation of radish 3-hydroxy-3-methylglutaryl-CoA reductase.

We propose the use of modified Lineweaver-Burk plots for the correct evaluation of Michaelis-Menten parameters in radioactive enzyme assays. A correction factor X for the translation of 1/S0 into 1/S is directly derived from the integrated Michaelis-Menten equation without the need of complicated calculations. In practice, this approach is favorably combined with an isotope dilution method which enhances the reliability of measurements at low substrate concentrations. The usefulness of the theoretical and practical approach is demonstrated in investigations of HMG-CoA reductase present in membrane fractions isolated from radish seedlings. The enzyme in the two main membrane fractions obtained by centrifugation at 16000 X g ( P16000 ) and at 105000 X g ( P105000 ) appears to be independently regulated by phytochrome and by phytohormones. Whereas active phytochrome decreases the apparent V of HMG-CoA reductase in the P105000 without affecting the Km, it increases the apparent Km in the P16000 . Kinetin treatment also results in a higher apparent Km of the enzyme in the P16000 fraction. Gibberellic acid and indoleacetic acid did not exhibit such a clear effect.

Localization of prenylquinones in the envelope of spinach chloroplasts.

The isolated and purified chloroplast envelope of spinach leaves contains, besides carotenoids, several prenylquinones as basic constituents: plastoquinone-9, phylloquinone K1, alpha-tocoquinone and the chromanol, alpha-tocopherol. The relative quinone and carotenoid composition of the envelope differs distinctively from that of the thylakoid membranes. The possible role of prenylquinones in metabolic envelope activities and the mediator function of the envelope in prenylquinone biosynthesis are discussed.

Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway.

Isopentenyl diphosphate (IPP) is the biological C5 precursor of isoprenoids. By labeling experiments using [1-(13)C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound isoprenoids (beta-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.

Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase of Arabidopsis thaliana.

Various plant isoprenoids are synthesized via the non-mevalonate pathway of isopentenyl diphosphate formation. In this pathway, 1-deoxy-D-xylulose 5-phosphate (DOXP), the first intermediate, is transformed to 2-C-methyl-D-erythritol 4-phosphate (MEP) by an enzyme which was recently cloned from Escherichia coli. In order to find a plant homologue of this 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) we cloned a cDNA fragment from Arabidopsis thaliana which has high homology to the E. coli DXR. By expression of this fragment in E. coli we could demonstrate that it encodes a protein which transforms DOXP to MEP. The antibiotic fosmidomycin specifically inhibits this DXR enzyme activity.

Incorporation of 1-deoxy-D-xylulose into isoprene and phytol by higher plants and algae.

In further substantiating the novel mevalonate-independent pathway for isoprenoid biosynthesis, which generates isopentenyl diphosphate (IPP) via 1-deoxy-D-xylulose-5-phosphate, labeling experiments with 1-[2H(1)]deoxy-D-xylulose were performed with various higher plants and algae: efficient incorporation was observed into isoprene emitted by Populus, Chelidonium, and Salix, into the phytol moiety of chlorophylls in a red alga (Cyanidium), in two green algae (Scenedesmus, Chlamydomonas), and a higher plant (Lemna). By contrast, 13C-mevalonate applied was incorporated into isoprene and phytol to a much lower extent or not at all. This demonstrates that this '1-deoxy-D-xylulose-5-phosphate pathway' for biosynthesis of plastidic isoprenoids is widely distributed in photosynthetic organisms.

The stress concept in plants: an introduction.

The current concept of stress in plants has been well developed over the past 60 years. Any unfavorable condition or substance that affects or blocks a plant's metabolism, growth, or development is regarded as stress. Vegetation stress can be induced by various natural and anthropogenic stress factors. One has to differentiate between short-term and long-term stress effects as well as between low-stress events that can be partially compensated for by acclimation, adaptation, and repair mechanisms, on the one hand, and strong stress or chronic stress events causing considerable damage that may eventually lead to cell and plant death, on the other hand. Some essential stress syndrome responses of plants are summarized in a unifying stress concept. The major abiotic, biotic, and anthropogenic stressors are listed. Some stress tolerance mechanisms are mentioned. Stress conditions and stress-induced damage in plants have so far been detected using the classical ecophysiological field methods as well as point data measurements of particular chlorophyll fluorescence parameters and of reflectance spectra. The novel laser-induced high-resolution fluorescence imaging technique, which integrates chlorophyll and blue-green fluorescence, marks a new standard in the detection of stress in plants.

The non-mevalonate isoprenoid biosynthesis of plants as a test system for new herbicides and drugs against pathogenic bacteria and the malaria parasite.

Higher plants and several photosynthetic algae contain the plastidic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate pathway (DOXP/MEP pathway) for isoprenoid biosynthesis. The first four enzymes and their genes are known of this novel pathway. All of the ca. 10 enzymes of this isoprenoid pathway are potential targets for new classes of herbicides. Since the DOXP/MEP pathway also occurs in several pathogenic bacteria, such as Mycobacterium tuberculosis, and in the malaria parasite Plasmodium falciparum, all inhibitors and potential herbicides of the DOXP/MEP pathway in plants are also potential drugs against pathogenic bacteria and the malaria parasite. Plants with their easily to handle DOXP/MEP-pathway are thus very suitable test-systems also for new drugs against pathogenic bacteria and the malaria parasite as no particular security measures are required. In fact, the antibiotic herbicide fosmidomycin specifically inhibited not only the DOXP reductoisomerase in plants, but also that in bacteria and in the parasite P. falciparum, and cures malaria-infected mice. This is the first successful application of a herbicide of the novel isoprenoid pathway as a possible drug against malaria.

Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors.

The essential steps of the novel non-mevalonate pathway of isopentenyl diphosphate and isoprenoid biosynthesis in plants are described. The first five enzymes and genes of this 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway are known. The herbicide fosmidomycin specifically blocks the second enzyme, the DOXP reductoisomerase. The DOXP/MEP pathway is also present in several pathogenic bacteria and the malaria parasite. Hence, all herbicides and inhibitors blocking this novel isoprenoid pathway in plants are also potential drugs against malaria and diseases caused by pathogenic bacteria.

[Distribution and relative concentrations of lipophilic plastid quinones in green plants]. / Verbreitung und relative Konzentration der lipophilen Plastidenchionone in grünen Pflanzen.

The distribution of lipophilic plastid quinones was investigated in green plants of different systematic position. The relative concentrations of plastid quinones, carotenoids and chlorophylls are presented. 1. The lipophilic plastid quinones (plastoquinone 45, α-tocoquinone, α-tocopherol and vitamin K1) have ubiquitous distribution in the photosynthetic apparatus of higher plants and of red, green, brown and bluegreen algae. The photoautotrophic bacteria and the fungi do not contain any of the plastid quinones. 2. The relative concentrations of lipophilic plastid quinones and carotenoids lie within certain standard concentration limits, which are similar for plants of different systematic position. Marked deviations from these concentrations are found in strongly sun-exposed leaves of trees and shrubs, in the older leaves of perennial plants and in the leaves of aurea-variegated plants. 3. Plastinoquinone 45 and α-tocopherol are present in all plants in higher concentrations than α-tocoquinone and the naphthoquinone vitamin K1. In most plants plastoquinone 45 occurs to 50-70% in the reduced form plastoquinol. The lipophilic redox system α-tocoquinone/tocopherol is present in general to 80-90% in the reduced form (α-tocopherol).

[Plastoglobuli and lipoquinone content of chloroplasts from Cereus peruvianus (L.) Mill]. / Plastoglobuli und Lipochinongehalt der Chloroplasten von Cereus peruvianus (L.) Mill.

The chloroplasts in the assimilation parenchym of Cereus stems possess numerous osmiophilic plastoglobuli, the size and frequency of which increase with increasing age of chloroplasts. Parallel to this there occurs an augmentation of the plastid quinone level (3 fold) and the carotenoid level (about 1.5 fold). Thus the chloroplasts of the stem tissue from Cereus also show a correlation between lipoquinone content and plastoglobuli size, which has been found before in the chloroplasts of several leaf tissues (LICHTENTHALER, 1969). An increase in the carotenoid levels has not, however, been found in chloroplasts from leaf tissues.It is assumed that the excess lipids of Cereus chloroplasts (lipoquinones and carotenoids) are deposited in the osmiophilic plastoglobuli.

[The fine structure of chromoplasts from plasmochromous perigon-leaves of Tulipa]. / Die Feinstruktur der Chromoplasten in plasmochromen Perigon-Blättern von Tulipa.

The chromoplasts of yellow perigon leaves of Tulipa derive from the young chloroplasts present in the bud stage. During chromoplast development and thylakoid breakdown many large plastoglobuli (diameter 80-360 nm) are formed. The function of the petal plastoglobuli as stores for the chromoplast lipids such as plastidquinones and carotenoids is discussed.

[The localization of plastidquinones and carotenoids in the chromoplasts of petals from sarothamnus scoparius (L.) wimm ex koch]. / Die Lokalisation der Plastidenchinone und Carotinoide in den Chromoplasten der Petalen von Sarothamnus scoparius (L.) Wimm ex Koch.

1. The yellow petals of Sarothamnus are colored by chromoplasts which contain free xanthophylls and carotenoid esters. During development of chromoplasts from young chloroplasts the secondary carotenoids are formed together with plastidquinones of the benzoquinone-type (mainly α-tocopherol). 2. The formation of secondary carotenoids already starts in the green petals of the buds. Their main synthesis however, proceeds parallel with a partial destruction of chlorophyll and thylakoids. The secondary carotenoids are accumulated predominantly in the plastids of the epidermal and subepidermal cells. Concurrently the number of plastoglobuli is strongly increased. 3. The isolated plastoglobuli show the same size distribution (40-200 nm) as in situ on the electromicrograms. They contain both lipid classes, carotenoids and plastidquinones. Most of the carotenoid-rich Sarothamnus-plastoglobuli are much less electron dense than the plastoglobuli of green leaf tissue, which contain only traces of carotenoids. The general function of plastoglobuli in chromoplasts as stores for carotenoids and plastidquinones is discussed.

Studies on the constancy of the blue and green fluorescence yield during the chlorophyll fluorescence induction kinetics (Kautsky effect).

Blue (F450) and green (F530) leaf fluorescence were studied together with the red chlorophyll fluorescence (emission maxima F690 and F735) during light-induced chlorophyll fluorescence induction kinetics (Kautsky effect) in predarkened leaves of wheat (Triticum aestivum L.) and soybean (Glycine max L.). The intensity of the red chlorophyll fluorescence decreased from maximum fluorescence Fm to steady-state fluorescence Fs, and the fluorescence ratio F690/F735 decreased by about 10% from Fm to Fs. However, blue and green fluorescence intensities remained constant throughout the measuring time. Consequently, the ratio of blue to red fluorescence (F450/F690) increased during chlorophyll fluorescence induction kinetics, whereas the ratio of blue to green fluorescence (F450/F530) remained unchanged within the same period. The knowledge of these ratios will be a prerequisite for the interpretation of remote sensing data from terrestrial vegetation.

Mechanisms of inhibition by mevinolin (MK 803) of microsome-bound radish and of partially purified yeast HMG-CoA reductase (EC.1.1.1.34).

1) In kinetic studies, mevinolin proved to be a highly specific inhibitor of partially purified yeast HMG-CoA reductase (Ki = 3.5 nM towards HMG-CoA) and of microsomal HMG-CoA reductase from etiolated radish seedlings (Ki = 2.2 nM). At low concentrations of NADPH, the inhibitor counteracts the sigmoidal response of plant HMG-CoA reductase activity towards the cosubstrate. At higher concentrations of NADPH, the inhibition pattern is of non-competitive type. 2) Our results are extensively compared with that obtained by the use of animal tissue and yeast as an enzyme source in order to discuss model systems probably valid to evaluate properties and regulation of plant as well as yeast HMG-CoA reductase.

[Plastoglobuli in different types of plastids from Allium cepa L]. / Plastoglobuli in Verschiedenen Differenzierungsstadien der Plastiden Bei Allium cepa L.

The occurence and size of plastoglobuli were studied in the different types of plastids from Allium cepa. The function of plastoglobuli as a reservoir for lipoquinones is discussed. 1. The white turgescent leaves of the onion contain chloroplasts in the cells close to the vascular bundles. These chloroplasts show the usual thylakoid arrangement and possess numerous plastoglobuli (av. diameter 65 nm). There also exist plastid-like components with concentrically arranged membranes and osmiophilic lipid inclusions (av. diameter 200 nm). 2. The chloroplasts in green sprouting leaves contain less and smaller plastoglobuli (diameter ca. 45 nm). 3. The plastoglobuli from leucoplasts possess the same size as plastoglobuli from chloroplasts in the onion leaves. The leucoplasts of the epidermis cells contain, however, less plastoglobuli than leucoplasts of the onion mesophyll. 4. The lipoquinone content in chloroplasts from green parts of the white turgescent onion leaves is much higher than in chloroplasts from green sprouting leaves. The concentrations of plastoquinone 45 are 25 X, of \ga-tocopherol 21 X, of \ga-tocoquinone 3,7 X and of vitamin K1 2,5 X higher with reference to chlorophyll a. 5. There is a direct correlation between lipoquinone and plastoglobuli content of plastids.

The influence of continuous far-red and white light on prenyl chain synthesis in plastids of Raphanus seedlings.

The rate of prenyl chain accumulation (C40 carotenoids; C45 in plastoquinone-9; C20 phytyl in chlorophylls, α-tocopherol and vitamin K1) in plastids of etiolated radish seedlings (Raphanus sativus L.) is determined in continuous darkness and after far-red and white light treatment. Continuous far-red light (active phytochrome P fr ) stimulates the synthesis of all prenyl chains, but has no or only little effect on the dark pattern of the prenyl chain formation. White light enhances the accumulation of prenyl chains to a much higher degree than does far-red light. By a particularly strong promotion of the accumulation of phytyl chains, which are incorporated into chlorophyll, white light changes the percentage composition of prenyl chains to that of chloroplasts.

Mevinolin: a highly specific inhibitor of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase of radish plants.

The fungal metabolite mevinolin, known to be a potent hypocholesterolemic agent, exerts in vitro a strong inhibitory effect on microsomal HMG-CoA reductase from etiolated radish seedlings at a concentration of about three magnitudes lower than the Km towards the natural substrate (S)-HMG-CoA (I50 = 2.5 x 10(-9)M). Beside this, mevinolin significantly inhibits the root elongation of radish as well as of wheat seedlings already at low concentrations of 10 to 100 ppb (= 2.5 x 10(-8) to 2.5 x 10(-7)M).

Incorporation of (14)CO 2 in prenylquinones of Chlorella pyrenoidosa.

Incorporation and release of (14)C-label in prenylquinones of Chlorella was investigated under steady state conditions. After one hour of (14)CO2-photosynthesis all plastid quinones investigated were labeled. The highest label was found in phylloquinone (18%) while α-tocopherol exhibits the lowest label (0.38%). Among the plastoquinones, plastohydroquinone-9 shows a higher labeling degree (5.1%) and a faster labeling kinetic than plastoquinone-9 (1.6%). After replacement of (14)CO2 against (12)CO2 the total radioactivity in plastohydroquinone-9, α-tocopherol and phylloquinone decreases but in α-tocoquinone and plastoquinone-9 proceeds further. From this labeling kinetic we conclude, that newly synthesized [(14)C]α-tocopherol molecules are converted to [(14)C]α-tocoquinone and [(14)C]plastohydroquinone-9 molecules to [(14)C]plastoquinone-9. From their (14)C-incorporation kinetic half-lives could be calculated for all prenylquinones in the same ranges as previously found for the chlorophylls and carotenoids (Grumbach et al., 1978). Half-lives are shorter in plastohydroquinone-9 (30 min) and plastoquinone-9 (40 min) than in phylloquinone (55 min), α-tocoquinone (50 min) and α-tocopherol (220 min). This means that all prenyl-lipids such as chlorophyll a, α-and ß-carotene, plastohydroquinone-9 and plastoquinone-9 which are more directly involved in the process of photosynthesis are subject to a continuous and higher turnover than the xanthophyll and α-tocopherol. From the fast labeling kinetic and short half-lives of α-tocoquinone and especially phylloquinone with a labeling degree of 12% after one hour of (14)CO2 photosynthesis we suppose that perhaps these two prenylquinones are also involved in the photosynthetic activity of chloroplasts.