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Inhibition of the Non-Mevalonate L-Deoxy-D-Xylulose-5-Phosphate

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Inhibition of the Non-Mevalonate L-Deoxy-D-Xylulose-5-Phosphate Inhibition of the Non-Mevalonate l-Deoxy-D-xylulose-5-phosphate Pathway of Plant Isoprenoid Biosynthesis by Fosmidomycin Johannes Zeidler3, Jörg Schwender3, Christian Müller3, Jochen Wiesnerb, Claus Weidemeyerb, Ewald Beckb, Hassan Jomaab and Hartmut K. Lichtenthaler 3 a Botanical Institute II, University of Karlsruhe, D-76128 Karlsruhe, Germany b Molecular Biology, Institute of Biochemistry, Academic Hospital Centre, Justus-Liebig University, D-35392 Giessen, Germany Z. Naturforsch. 53c, 980-986 (1998); received October 2, 1998 Carotenoid Biosynthesis, Fosmidomycin, Isopentenyl Diphosphate, Isoprene, Non-Mevalonate IPP Pathway, Terpenoids Various bacterial and plastidic plant terpenoids are synthesized via the non-mevalonate l-deoxy-D-xylulose-5-phosphate (DOXP) pathway. The antibiotic and herbicidal compound fosmidomycin is known to inhibit growth of several bacteria and plants, but so far its mode of action was unknown. Here we present data which demonstrate that the DOXP pathway of isoprenoid biosynthesis is efficiently blocked by fosmidomycin. The results point to the DOXP reductoisomerase as the probable target enzyme of fosmidomycin. Introduction of plant isoprenoid biosynthesis - plastids and The alternative non-mevalonate 1-deoxy-D-xy- cytosol - possess two different IPP pathways: the lulose-5-phosphate (DOXP) pathway for isopren­ plastidic DOXP pathway and the cytosolic MVA oid formation is widespread in the plant kingdom pathway (Lichtenthaler, 1998; Schwender et al., (for review see Lichtenthaler et al., 1997a; Lichten­ 1997). In addition to higher plants, we also found thaler, 1998). It is also present in various bacteria among eukaryotic algae (heterokontophyta, rho- (Rosa Putra et al., 1998a). In higher plants, chloro- dophyta and chlorophyta) the formation of plas- plast-bound isoprenoids (carotenoids, phytol side- tid-bound phytol and carotenoids according to the chain of chlorophylls, nonaprenyl side-chain of alternative DOXP pathway (Schwender et al., plastoquinone-9) are formed via the DOXP path­ 1996; Disch et al., 1998). Isoprene, as the simplest way (Lichtenthaler et al., 1997b; Schwender et al., plant isoprenoid and as a close derivative of IPP, 1997; Arigoni et al., 1997). Also the formation of is synthesized and emitted by many plants at high lycopene in chromoplasts of ripening tomato fruits light conditions and high temperatures (Sharkey, proceeds via this alternative DOXP pathway 1996). Isoprene is synthesized according to the (Schwender and Lichtenthaler, 1998). The sterols DOXP pathway (Schwender et al., 1997; Zeidler of higher plants, in turn, are formed and accumu­ etal., 1997). lated in the cytosolic cell compartment via the The DOXP pathway starts with the formation classical mevalonate (MVA) pathway (Lichten­ of l-deoxy-D-xylulose-5-phosphate (DOXP) from thaler et al., 1997a, b). The two cell compartments glyceraldehyde-3-phosphate and pyruvate by the enzyme DOXP synthase (see Lichtenthaler, 1998 and references therein) (Fig. 1). DOXP is subse­ Abbreviations: a and b , chlorophyll a and b; a/b, weight quently transformed to 2-C-methyl-D-erythritol 4- ratio of chlorophyll a to 6; c, ß-carotene; dw, dry weight; phosphate by an intramolecular C -C skeleton re­ DOX, 1-deoxy-D-xylulose; DOXP, l-deoxy-D-xylulose-5- arrangement (Fig. 1). This is indicated by the spe­ phosphate; GA-3-P, glyceraldehyde-3-phosphate; cific incorporation of 2 H-, 1 3 C- or 1 4 C-labeled 1- IpOHA. N-isopropyl-N-hydroxyoxamate; IPP, isopen­ tenyl diphosphate; KARI. ketol acid reductoisomerase; deoxy-D-xylulose into isoprene and phytol MVA, mevalonic acid; x, xanthophylls; x+c, total carot­ (Zeidler et al., 1997; Schwender et al., 1997), into enoids. ß-carotene (Arigoni et al., 1997), into 2-C-methyl- Reprint requests to Prof. Dr. Dr. h. c. H. K. Lichten­ D-erythritol of plants (Sagner et al., 1998), and into thaler. Fax: +49 721 608 4874. bacterial ubiquinone (Rosa Putra et al., 1998b). In E-mail: [email protected] addition, a DOXP reductoisomerase which trans- 0939-5075/98/1100-0980 $ 06.00 © 1998 Verlag der Zeitschrift für Naturforschung, Tübingen ■ www.znaturforsch.com. N J. Zeidler et al. ■ Inhibition of Plant Isoprenoid Biosynthesis by Fosmidomycin 981 Pyruvate + GA-3-P | DOXP synthase OH v V .°p O OH Fig. 2. Chemical structure of fosmidomycin, a specific in­ 1-Deoxy-D-xylulose-5-P (DOXP) hibitor of the non-mevalonate l-deoxy-D-xylulose-5- phosphate (DOXP) pathway of isoprenoid biosynthesis. I DOXP reductoisomerase * (+ NADPH) son, 1987; Kamuro et al., 1988). However, its mode of action has so far not been evaluated. OP For this reason we tested, if fosmidomycin can OH OH inhibit the plants’ biosynthesis of plastidic isopren- 2-C-Methyl-D-erythritol-4-P oids, such as carotenoids, phytol (side-chain of l chlorophylls), and isoprene which are formed via the novel, alternative, non-mevalonate DOXP J pathway of IPP biosynthesis (Lichtenthaler, 1998). Materials and Methods OPP Plant material Isopentenyl diphosphate (IPP) Barley grains ( Hordeum vulgare L., cv. Alexis) Fig. 1. Biosynthesis of the isoprenoid precursor isopen­ were germinated in the dark. Tomato plants ( Ly- tenyl diphosphate (IPP) from pyruvate and GA-3-P via copersicon esculentum L., cv. Sweet 100) were the non-mevalonate DOXP pathway. The C-atoms 1, 2 and 4 of IPP derive from GA-3-P and the C-atoms 3 and grown in the greenhouse, and duckweed plantlets 5 from pyruvate. (Lemna gibba L.) in continuous light (100 [imol m~2 s_1) on a liquid mineral medium (Lichten­ thaler et al., 1997b). forms DOXP to 2-C-methyl-D-erythritol-4-phos- Greening of etiolated barley leaves phate was cloned in E. coli (Takahashi et al., 1998). It appears that the DOXP reductoisomerase reac­ Primary etiolated leaves of 5-6 d old barley tion may have a mechanism similar to the ketol seedlings were cut and placed with the cut ends in acid reductoisomerase (KARI), which operates in the dark into a mineral medium (Lichtenthaler et the biosynthesis of valine, leucine and isoleucine al., 1997b) with different concentrations of fosmi­ (Aulabaugh and Schloss, 1990). Both enzymes cat­ domycin. The inhibitor was taken up by transpira­ alyze a C -C skeleton rearrangement followed by tion which was promoted by a continuous air a NADPH-dependent reduction step (Aulabaugh stream (ventilator). After a 5 h dark period for up­ and Schloss, 1990; Takahashi et al., 1998). take of the fosmidomycin the leaves were exposed In our search for inhibitors of the plants’ DOXP to light ( 1 0 0 p,mol m - 2 s-1) for a 18 h greening pathway we were looking for analogues to KARI period. inhibitors, such as N-isopropyl-N-hydroxyoxamate (IpOHA) (Aulabaugh and Schloss, 1990; Biou et Ripening of tomato fruits al., 1997). Fosmidomycin (Fig. 2), an antibiotic Small tomato fruits were harvested just at the produced by Streptomyces lavendulae (Okuhara et beginning of ripening when the change of colour al., 1980), had formerly been shown to affect iso­ from green to red was first visible. Into each to­ prenoid biosynthesis in several bacteria (Shigi, mato fruit (5-6 g fresh weight) 10-30 ^tl of a 50 1989) and also to have herbicidal activity (Patter- m M solution of fosmidomycin was injected. The 982 J. Z eidler et al. • Inhibition of Plant Isoprenoid Biosynthesis by Fosmidomycin control fruits with much lycopene accumulation The reaction proceeded at 37 °C. At the begin­ and the treated tomato fruits were extracted af­ ning and after 5 min 10 [xl of the solution were ter 1 0 d. taken and the reaction stopped by 1 0 jj,l ice cold acetone. l-Deoxy-D-xylulose-5-phosphate and pyr­ Growth of duckweed plantlets uvate were separated by TLC (/ 7 -propyl alcohol/ ethylacetate/H20 6:1:3; Lois et al., 1998). Bands Plantlets of Lemna gibba L. were grown for 6 d (DOXP Rf 0.35, pyruvate Rf 0.59) were scraped on a liquid mineral medium (Lichtenthaler et al., off and the radioactivity was counted in a liquid 1997b) under continuous light (100 [imol m~ 2 s~') scintillation counter (Packard 2000 CA). Concen­ 1 0 - 4 m, without (controls) and with fosmidomycin ( trations of 1 0 “ 4 m and 1 0 “ 3 m fosmidomycin were 5 x l0 - 5 m , 2.5x l 0 ~ 5 m ) in the nutrition solution. applied in the enzyme test. Determination of chlorophylls and carotenoids Synthesis of fosmidomycin After grinding of plant material and cold extrac­ Fosmidomycin, 3-(N-formyl-N-hydroxyamino)- tion with acetone, chlorophylls a and b as well as propylphosphonate, was synthesized using a com­ total carotenoids x+c were determined spectro- bination of the protocols reported before (see photometrically (Lichtenthaler, 1987). Kamiya et al., 1980; Öhler and Kanzler, 1995). D e­ tailed protocols of the synthesis can be obtained Isoprene emission from one of the authors, H. Jomaa, Giessen. Leaves of Populus nigra L., Platanus x acerifolia Results and Discussion and Chelidonium majus L. were taken from plants growing on the campus of the University of Karls­ Since many algae possess the DOXP pathway ruhe. Isoprene emission was quantified using a for the synthesis of plastidic isoprenoid pigments newly developed UV-cuvette test system (Zeidler (Schwender et al., 1997; Disch et al., 1998), we and Lichtenthaler, 1998). Freshly cut rectangular tested the effect of fosmidomycin in a red alga. leaf pieces were placed at one inner side of a When applied to Cyanidium caldarium fosmido­ quartz cuvette containing 0.5 ml of either water or mycin fully inhibited cell multiplication and pig­ ment accumulation at a level of 5xl0 - 5 m and an aqueous solutions of fosmidomycin or 1 -deoxy- D-xylulose. The leaf pieces were illuminated in the 5 x l0 - 4 m (data not shown). Also in higher plants the formation of chlorophylls and carotenoids is closed cuvette with white light ( 1 0 0 0 |imol photons blocked by fosmidomycin as shown below.
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