QUARTERLY Review Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-Cmethyl-D-erythritol

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.


INTRODUCTORY REMARQUES
Isoprenoids are a broad group of natural compounds with carbon skeleton built of branched C 5 isoprenoid units.Isoprenoids are widespread among living organisms, both eukaryotes and prokaryotes, functionally important for many aspects of cell metabolism, and they influence also membrane structure and function.Many important biological roles of numerous isoprenoids have been described in: photosynthesis (carotenoids, chlorophylls, plastoquinone), respiration (ubiquinone), hormonal regulation of metabolism (sterols), regulation of growth and development (giberellic acid, abscisic acid, brassinosteroids, cytokinins, prenylated proteins), defense against pathogen attack, intracellular signal transduction (Ras proteins), vesicular transport within the cell (Rab proteins) and as coenzymes (dolichols).Several isoprenoids are also known to influence membrane structure (sterols, dolichols, carotenoids etc.) (Sacchettini & Poulter, 1997;Bach et al., 1999).
The early steps of isoprenoid biosynthesis have been studied first in vitro using cell-free homogenates obtained from rat liver and yeast cells by Bloch and Lynen (Chaykin et al., 1958, Lynen et al., 1958).The specific precursor of all isoprenoids, mevalonate (MVA) was found to be synthesized by the condensation of three acetyl-CoA molecules via acetoacetyl-CoA and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), yielding after phosphorylation and decarboxylation isopentenyl diphosphate (IPP).All the enzymes involved in the MVA pathway were isolated and studied in many animal and plant systems, however some results obtained with the labeled precursor -[ 3 H]mevalonate were difficult to explain.Current biosynthetic evidence cames from the application of 13 C-labeled precursors and subsequent exact NMR analysis of the position of the 13 C-atoms incorporated within the isoprenoid carbon skeleton.These results suggest the existence of an MVA-independent pathway for IPP formation in bacteria (Rohmer et al., 1993), green algae (Lichtenthaler et al., 1995;Schwender et al., 1996) and higher plants (Lichtenthaler et al., 1997;Schwender et al., 1997) and also in the malaria parasite Plasmodium falciparum (Jomaa et al., 1999).On the basis of labeling patterns (Fig. 1) of isoprenoid carbon skeletons derived from 13 C-labeled metabolites of glycolysis as well as [ 13 C]acetate, it has been shown that IPP in chloroplasts is synthesized from pyruvate and glyceraldehyde-3-phosphate and not from MVA.

BIOSYNTHESIS OF THE ISOPRENOID PRECURSOR IPP VIA THE NOVEL, DOXP/MEP PATHWAY
As suggested by 13 C-labeling experiments, the pathway starts with the addition of a pyruvate-derived C 2 -unit to glyceraldehyde-3phosphate (GA-3-P) (Rohmer et al., 1993;Schwender et al., 1996) (Fig. 2).This was supposed to take place in a transketolase-type enzymatic reaction.After TPP-catalyzed decarboxylation of pyruvate, the TPP-bound acetaldehyde is added to the carbonyl group of GA-3P yielding 1-deoxy-D-xylulose 5-phosphate (DOXP) as the first intermediate (Schwender et al., 1997;Arigoni et al., 1997).The starting enzyme of this pathway is 1-deoxy-D-xylulose 5-phosphate synthase (DXS).The dxs gene has been cloned from several higher plants (Lange et al., 1998;Bouvier et al., 1998), Escherichia coli (Lois et al., 1998;Sprenger et al., 1997), green alga -Chlamydomonas (Lichtenthaler, 1999) and from a strain of Streptomyces (Kuzuyama et al., 2000a).The enzyme requires thiamine diphosphate and divalent cations such as Mg 2+ or Mn 2+ for its activity (Sprenger et al., 1997;Bouvier et al., 1998).Based on sequence data from nucleic acid data bases it can be concluded that the DXS-like sequences are highly conserved in evolution.DXS-like gene (CLA1) found in Arabidopsis thaliana (Mandel et al., 1996) is supposed to be a single copy gene regulated by light.The mutation of the CLA1 impairs the proper development of chloroplasts, arresting these organelles at an early stage of development.In addition, 1-deoxy-D-xylulose is also an intermediate in the biosynthesis of coenzymes thiamine and pyridoxal phosphate (Julliard & Douce, 1991) (Fig. 2).The second enzymatic step -a C-C-skeleton rearrangement and reduction of 1-deoxy-D-xylulose-5-phosphate to 2-C-methyl-D-erythritol 4-phosphate (MEP) is catalyzed by dxr gene product, namely DOXP-reductoisomerase (DRI or DXR) in the presence of NADPH and Mn 2+ (Fig. 2).The enzyme transforming DOXP to MEP has been charac-terized in E. coli (Kuzuyama et al., 2000b).A cloning strategy was also developed for isolation of the gene encoding a plant homolog of this enzyme from peppermint (Lange & Croteau, 1999), A. thaliana (Schwender et al., 1999), blue-green alga Synechocystis (Proteau, 1998) and the parasite Plasmodium falciparum (Jomaa et al., 1999).Unlike the microbial reductoisomerase, the plant ortholog encodes a preprotein bearing an N-terminal plastidial transit peptide that directs the enzyme to the plastids.It was found that DXR activity was strongly and specifically inhibited by fosmidomycin, an antibiotic possessing formyl and phosphonate groups in the molecule (Kuzuyama et al., 1998).It was also shown that fosmidomycin inhibits the biosynthesis of carotenoids and chlorophylls in greening leaves, as well as the isoprene emission (Zeidler et al., 1998).Recently it has been reported by Jomaa et al. (1999) that fosmidomycin and its derivative inhibit DXR from P. falciparum and that this inhibitor cured mice infected with the rodent malaria parasite P. vickei.Thus, this inhibitor can be possibly effective in chemotherapy of malaria.

OCCURRENCE OF THE MEP PATHWAY IN DIFFERENT ORGANISMS
The mevalonate-independent pathway for IPP synthesis was first discovered by Flesch & Rohmer (1988) in their studies on biosynthesis of hopanoids (i.e.pentacyclic triterpenic sterol surrogates of different bacterial species) using [ 13 C]acetate.This biosynthetic scheme for IPP formation was later found in E. coli, Alicyclobacillus acidocaldarius, Methylobacterium organophilum, Zymomonas mobilis (Flesch & Rohmer, 1988;Rohmer et al., 1993).Several observations showed that in a few Archaebacteria (Pyrococcus horikoshii, Methanococcus jannaschii, Methanobacterium thermoautotrophicum) only the mevalonate pathway is operating (Lichtenthaler et al., 2000).In most bacterial strains only one of the two IPP biosynthetic pathways seems to appear.Helicobacter pylori may be an exception since its genome contains also a HMG-CoA reductase ortholog (Lichtenthaler et al., 2000).
The 13 C-labeling studies showed that in red algae (Cyanidum), chrysophyte (Ochromonas) and oxygenic photosynthetic blue-green bacteria (Synechocistis) cytoplasmic sterols are formed via the mevalonate pathway.In contrast, in all cases the plastid-bound isoprenoids, such as phytol, b-carotene, lutein and the side chain of plastoquinone-9 exhibited the MEP-pathway labeling pattern (Schwender et al., 1997).Further studies on the green algae Scenedesmus obliquus, Chlorella fusca and Chlamydomonas reinhardtii showed that not only the plastidic isoprenoids but also their cytosolic sterols and mitochondrial ubiquinones (Scenedesmus) (Schwender et al., 1996;Disch et al., 1998b) were labeled via the DOXP/MEP pathway.In addition, the labeled MVA was not incorporated into the sterols of these green algae (Schwender et al., 1997) indicating that these organisms have lost during evolution, or they never possessed, the classical mevalonate pathway.However, it is also possible that, a low rate of biosynthesis via the mevalonate pathway might have been undetected by the 13 C-labeling technique (Lichtenhaler et al., 1997).In the case of Euglena gracilis the plastidic phytol as well as the cytosolic ergosterol were labeled from [1-13 C]glucose and from 13 C-labeled mevalonate according to the pattern specific for the mevalonate pathway (Disch et al., 1998b).
Investigations with specific intermediates of both pathways such as [1-2 H]DOX and [2-13 C]MVA showed in Cyanidum, Ochromonas and Euglena that small amounts of not only [ 13 C]sterols but also [ 13 C]phytol could be formed from [2-13 C]MVA while [1-2 H]DOX was found to serve as the precursor of both phytol and ergosterol; this points to the possibility of exchange between the two pools of IPP of different biosynthetic origin (Lichtenthaler, 1998) (Fig. 3).
In higher plants the labeling experiments with 13 C-labeled mevalonate and 2 H-labeled 1-deoxy-D-xylulose, suggested that biosynthesis of IPP occurs at two sites.The first one is located in the cytoplasm operates via the mevalonate pathway, and the produced IPP is subsequently distributed to the endoplasmic reticulum and mitochondria for the formation of triterpenoids (including sterols), sesquiterpenoids and the prenyl chain of ubiquinone.The second site of IPP biosynthesis, located to the chloroplasts, produces via MEP pathway IPP for the formation of all isoprenoids involved in photosynthesis: phytol, plastoquinone, carotenoids (Schwender et al., 1997;Arigoni et al., 1997;Lichtenthaler et al., 1997).It is worth noting that these experiments were performed under very low light conditions.Additionally, in the study on tobacco, the experiments were carried out using a heterotrophically grown cell culture (BY-2) system devoid of functional chloroplasts (Disch et al., 1998a).
Simultaneously, labeling of PQ and UQ with [ 3 H]mevalonate was observed in etiolated and non-etiolated spinach seedlings pointing to MVA-dependent origin of IPP in these experimental conditions (Wanke et al., 2000).Recently it has been also found that polyisoprenoid alcohols occurring in the roots of Coluria geoides grown in vitro are formed from mevalonate (Skorupiñska-Tudek, K., unpublished).
The study of chloroplasts metabolism during early developmental stages performed by Heintze et al. (1994) showed that in immature chloroplasts from young spinach plants [2-14 C]acetate incorporation into chloroplastic isoprenoids and fatty acids was about five times higher than that of [2-14 C]pyruvate.These results indicate that the synthesis of IPP occurs via chloroplast mevalonate rather than the DOXP/MEP pathway.A further study confirmed the existence of chloroplastic mevalonate kinase, detected only after breaking of the chloroplasts (Preiss & Schultz, 1994).Evidence for nuclear genes encoding early steps of the mevalonate pathway enzymes localized in the endoplasmic reticulum (ER) but not in the plastids was obtained but it was difficult to detect their expression possibly because of its very low level at these developmental stages.Therefore Heintze et al. (1994) suggested that the genes encoding the chloroplastic mevalonate pathway enzymes are expressed only at an early stage of chloroplast development.(mevalonate pathway) and plastid (DOXP/MEP pathway) (Lichtenthaler, 1999, modified).
The labeling pattern of the phytyl side-chain of chlorophyll determined by incorporation of 2 H or 13 C-labeled acetates, glycerol or glucose in Heteroscyphus planus cells suggests simultaneous operation of the novel, DOXP/MEP and the mevalonate pathway (Nabeta et al., 1997).Additionally, reports were published on the existence of a plastidic, along with an ER associated, HMG-CoA reductase (Wong et al., 1982;Kim et al., 1996).These, sometimes contradictory results suggest the possibility that indeed two pathways of IPP formation are operating and their activation is dependent on physiological conditions or developmental stage of the cell.

COMPARTMENTATION OF IPP BIOSYNTHESIS IN HIGHER PLANTS AND ENDOSYMBIOTIC THEORY
In higher plants the MEP pathway derived IPP is used not only for the biosynthesis of isoprenoids involved in photosynthesis but also for the formation of the volatile hemiterpene isoprene (Schwender et al., 1997), taxol (Eisenreich et al., 1996) and marrubiin (Knöss et al., 1997).
Fast incorporation of 14 CO 2 into isoprene suggested that isoprene synthesis was related to the photosynthetic CO 2 fixation.In addition, isoprene synthase has been found in plastids.Further evidence came from labeling experiments using deuterium labeled DOX or its methyl glycoside.High incorporation rates of this intermediate into isoprene were found in poplar (Populus nigra), celandine (Chelidonium maius) and willow (Salix viminalis) (Zeidler et al., 1997).
According to the endosymbiotic theory, the plastid compartment is a heritage from photosynthetic prokaryotic ancestors.The fact that the phytol of chlorophyll a in the photosynthetic bacteria is also formed by the MEP pathway supports this theory.Thus, it appears that the plastids have maintained the original bacterial DOXP/MEP pathway of IPP biosynthesis during coevolution with the eukaryotic plant cells (Lichtenthaler et al. 2000).

COOPERATION BETWEEN TWO PATHWAYS OF IPP BIOSYNTHESIS IN HIGHER PLANTS
Several observations led to the suggestion that some exchange and cooperation between the two pathways does exist, but still it is not clear to what extent two different pools of IPP or other prenyl diphosphates such as geranyl diphosphate, farnesyl diphosphate or geranylgeranyl diphosphate are exchangeable (Fig. 3).Nabeta et al. (1997) studing the 13 C-labeling of chlorophyll and carotenoids found that cytosolic farnesyl diphosphate was transferred into plastid, where it was condensed with a DOXP derived IPP.The export of IPP or geranyl diphosphate from the plastids into the cytosol may also exist, as it was shown by inhibitor studies (Schwender et al., 1997).Several observations suggest the occurrence of at least some exchange.Three isoprene units were found to be labeled via the MVA pathway, and the fourth isoprene unit via the DOXP/MEP pathway upon labeling of the diterpene ginkgolide from [ 13 C]glucose (Lichtenthaler, 1999).In the liverwort Heteroscyphus, the first three isoprenic units of phytol showed some label from the applied [ 13 C]MVA, whereas the fourth unit was not labeled (Nabeta et al., 1997).Recent studies on chamomile also suggest that two cellular IPP pools might cooperate and exchange IPP or GPP (Adam & Zapp, 1998).As shown in these experiments, the first two C 5 -units of sesquiterpene molecules were derived from [ 13 C]glucose via the DOXP/MEP pathway, and the third C 5 -unit was labeled either by the DOXP or the MVA pathway.
Recent discoveries have contested the well established model of isoprenoid biosynthesis in bacteria and plants.The results discussed above indicate that both the classical, MVA and the novel, DOXP/MEP pathway are present within at least some, if not all, plant cells.It is the task for the future to establish the mechanisms of spatial and temporal modulation of the activity of the enzymes involved in these two parallel pathways leading to the formation of IPP.
We are grateful to Professor M. Rohmer and Professor T. Chojnacki for discussions and stimulation.

Figure 3 .
Figure 3. Compartmentation of the IPP and isoprenoid biosynthesis within plant cells between cytosol