Jörg Schwender

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 [1‐13C]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 (β‐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‐[2H1]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.

Inhibition of the Non-Mevalonate 1-Deoxy-ᴅ-xylulose-5-phosphate Pathway of Plant Isoprenoid Biosynthesis by Fosmidomycin

Abstract Various bacterial and plastidic plant terpenoids are synthesized via the non-mevalonate1-deoxy-ᴅ-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 reductoisom erase as the probable target enzyme of fosmidomycin.

Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids

Abstract. The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways: in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostigma viride the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-d-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.

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-ᴅ-xylulose 5- phosphate / 2-C-methyl-ᴅ-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.

A Novel Mevalonate-Independent Pathway for the Biosynthesis of Carotenoids, Phytol and Prenyl Chain of Plastoquinone-9 in Green Algae and Higher Plants

The acetate/mevalonate pathway, which provides IPP, had been investigated in the 50’s and 60’s in detail, and the universal occurrence of this pathway in animal and plant isoprenoid biosynthesis was generally accepted. However, some observations conflicted with this pathway. The biosynthesis of plastidic isoprenoids of higher plants (carotenoids, chlorophylls, plastoquinone-9) was not inhibited by mevinolin, a highly specific inhibitor of mevalonate formation (1, 2). In order to test if the plastidic isoprenoids of algae and higher plants are formed via the acetate/mevalonate pathway or via a different pathway some 13C-labelling experiments were carried out with two green algae and the higher plant Lemna. Here we show that in all three cases carotenoids and the prenyl side chains of chlorophylls and plastoquinone-9 are labelled via a new, bacterial and mevalonate-independent pathway of IPP biosynthesis (3, 4, 5).

The 1-deoxy-d-xylulose-5-phosphate Pathway for Biosynthesis of Carotenoids and Other Plastidic Isoprenoids

The biosynthesis of plastidic isoprenoids (carotenoids, phytol, plastoquinone-9) as well as cytosolic sterols and various terpenes seemed to have been established since around 1958 (see Review 1). From 14C-labeling studies it had been accepted that the photosynthetic plants and algae form their isoprenic C5-unit isopentenyl diphosphate (IPP) - as in animal systems and fungi - via the acetate/mevalonate (MVA) pathway (1, 2). Various observations on plastid isoprenoids were, however, not in agreement with this view. Thus, photosynthetically fixed 14CO2 was readily incorporated into plastidic isoprenoids (carotenoids, phytol, plastoquinone-9), whereas 14C-labeled acetate and MVA were rapidly incorporated into the cytosolic sterols, but only at low rates into the plastidic isoprenoids (see 1). In addition, mevinolin, a highly specific inhibitor of the HMG-CoA reductase, efficiently inhibited sterol accumulation, but did not affect the formation of carotenoids, phytol and plastoquinone-9 (3). After the detection of a novel IPP pathway in bacteria (4) we started in 1993 a re-investigation of the biosynthesis of plastidic isoprenoids in cooperation with the chemists M. Rohmer (Strasbourg) and F. W. Lichtenthaler (Darmstadt). Applying 13C- and 2H-labeling techniques, NMR spectroscopy and GC-MS analysis it was shown that green algae (chlorophyta), higher plants, and several other algae groups synthesize their plastidic isoprenoids including isoprene via the novel non-mevalonate 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway (1, 5–12). This novel DOXP pathway of IPP formation, which is obviously located in plastids, is reviewed here.

BIOSYNTHESIS OF STEROLS IN GREEN ALGAE (SCENEDESMUS, CHLORELLA) ACCORDING TO A NOVEL, MEVALONATE-INDEPENDENT PATHWAY

The biosynthesis of isopentenyl pyrophosphate (IPP), the biological precursor of isoprenoid substances, is known as the acetate mevalonate pathway. In the latter pathway the condensation of three acetyl-CoA, followed by two reduction steps (HMGCoA reductase), yields mevalonic acid which is converted to IPP. In higher plants, the biosynthesis of sterols occurs via mevalonic acid (1) and can be inhibited by mevinolin, a highly specific inhibitor of mevalonate formation (2). In contrast, in several green algae we could not find any inhibition effect of mevinolin on growth and multiplication of cells. Some 13C-labelling experiments in the green alga Scenedesmus (3) showed that the main sterol components are synthesized via a novel mevalonate-independent glyceraldehyde phosphate/pyruvate pathway of IPP biosynthesis first found in some eubacteria (4, 5). Here we show that this also applies to Chlorella.