A high-performance liquid chromatography method for the analysis of intermediates of the deoxyxylulose phosphate pathway

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 335 (2004) 235–243 www.elsevier.com/locate/yabio

A high-performance liquid chromatography method for the analysis of intermediates of the deoxyxylulose phosphate pathway Maja Raschke, Monika Fellermeier, Meinhart H. Zenk¤ Biozentrum der Universität Halle, Weinbergweg 22, D-06120 Halle (Saale), Germany Received 10 July 2004 Available online 28 October 2004

Abstract A sensitive and versatile ion pair radio high-performance liquid chromatography (HPLC) method for the investigation of the deoxyxylulose phosphate (DXP) pathway has been developed, allowing the simultaneous separation of phosphorylated, nonphosphorylated, and nucleotide moieties bearing intermediates. Moreover, this method addresses the problem of separating the isomers isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP). Because the majority of the intermediates of this isoprenoid pathway lack a chromophore, the combination with an on-line radiodetector provides a highly sensitive tool for their detection. Chromoplasts isolated from Capsicum annuum and Narcissus pseudonarcissus served as model systems for the testing of the analytical procedures after the application of radiolabeled precursors. This HPLC system, which represents an improvement in analytical methods developed for the analysis of the mevalonic acid pathway, should be easily adaptable to other plant and bacterial systems and should permit further elucidation of the regulatory mechanisms that control the Xow of intermediates through the DXP pathway and the coordination with related metabolic pathways. Moreover, the system can serve as an analytical tool in the screening for inhibitors of this pathway, allowing the development of new antibiotics as well as herbicides, because this pathway is absent in vertebrates.  2004 Elsevier Inc. All rights reserved. Keywords: Reversed-phase ion pair HPLC; Isoprenoid biosynthesis; Deoxyxylulose phosphate pathway; Chromoplast

Isoprenoids represent one of the largest, but also structurally and functionally one of the most diverse, groups of natural products that play important roles in all living organisms [1]. Despite their diversity, all isoprenoids are derived from two universal Wve-carbon precursors: isopentenyl diphosphate (IDP, 9)1 and its isomer dimethylallyl diphosphate (DMADP, 10). In plants, there are two biosynthetic routes to these precursors: *

the cytosolic mevalonic acid (MVA) pathway and the deoxyxylulose phosphate (DXP) pathway, which is localized in plastids (Fig. 1). The latter one, which also exists in many eubacteria but is absent in mammals, has long been overlooked. However, during the past few years, knowledge about this pathway has emerged rapidly, and the joint contribution of genomics and conventional biochemical and genetic approaches led to its

Corresponding author. Fax: +49 345 55 27 301. E-mail address: [email protected] (M.H. Zenk). 1 Abbreviations used: IDP, isopentenyl diphosphate; DMADP, dimethylallyl diphosphate; MVA, mevalonic acid; DXP, 1-deoxy-D-xylulose 5phosphate; D-GAP, D-glyceraldehyde 3-phosphate; MEP, 2C-methyl-D-erythritol 4-phosphate; CDP–ME, 4-diphosphocytidyl-2C-methyl-D-erythritol; CMS, CDP–ME synthase; CMK, CDP–ME kinase; cMEDP, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; MCS, cMEDP synthase; CDP–MEP, 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate; HMBDP, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate; HDS, HMBDP synthase; IDDS, IDP/DMADP synthase; IDI, IDP/DMADP isomerase; HPLC, high-performance liquid chromatography; TBAS, tetra-n-butylammonium hydrogen sulfate; TLC, thin-layer chromatography; DTT, dithiothreitol; IP, isopentenyl phosphate. 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.09.013

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Fig. 1. The DXP pathway of isoprenoid biosynthesis. Enzymes of the pathway are labeled in bold with the corresponding genes in parentheses. DXS, DXP synthase; DXR, DXP reductoisomerase; CMS, CDP– ME synthase; CMK, CDP–ME kinase; MCS, cMEDP synthase; HDS, HMBDP synthase; IDDS, IDP/DMADP synthase; IDI, IDP/ DMADP isomerase. Precursors and intermediates of the DXP pathway are numbered in bold: 1, pyruvate; 2, D-glyceraldehyde 3phosphate (D-GAP); 3, 1-deoxy-D-xylulose 5-phosphate (DXP); 4, 2C-methyl-D-erythritol 4-phosphate (MEP); 5, 4-diphosphocytidyl2C-methyl-D-erythritol (CDP–ME); 6, 4-diphosphocytidyl-2C-methylD-erythritol 2-phosphate (CDP–MEP); 7, 2C-methyl-D-erythritol 2,4-cyclodiphosphate (cMEDP); 8, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP); 9, isopentenyl diphosphate (IDP); 10, dimethylallyl diphosphate (DMADP).

complete elucidation in Escherichia coli and, for most steps, also in plants [2–4]. The initial step involves the condensation of pyruvate (1) with D-glyceraldehyde 3phosphate (D-GAP, 2) to aVord DXP (3) [5], which is converted to 2C-methyl-D-erythritol 4-phosphate (MEP, 4) [6]. The next three steps, catalyzed by the enzymes 4diphosphocytidyl-2C-methyl-D-erythritol (CDP–ME) synthase (CMS), CDP–ME kinase (CMK), and 2Cmethyl-D-erythritol 2,4-cyclodiphosphate (cMEDP) synthase (MCS), led to the formation of cMEDP (7) via CDP–ME (5) and its 2-phosphate (CDP–MEP, 6) [7–12]. The protein speciWed by the ispG gene [13,14], (E)-4hydroxy-3-methylbut-2-enyl diphosphate (HMBDP) synthase (HDS), catalyzes the reductive transformation of the cyclic diphosphate into HMBDP (8). In the Wnal step, HMBDP (8), catalyzed by isopentenyl/dimethylallyl diphosphate synthase (IDDS), is converted to IDP (9) as well as DMADP (10) [15–17], thereby representing the branch point of the DXP pathway (Fig. 1). In contrast to bacteria, where it has been shown that the IDP/ DMADP isomerase (IDI) is dispensable and that the genomes do not contain open reading frames for this isomerase, with the exception of E. coli and Mycobacterium tuberculosis [18], plants express this enzyme and it localizes to the cytosol, mitochondria, and plastids [19]. The importance of this enzyme within the DXP pathway in plants was demonstrated recently by virus-induced gene silencing in Nicotiana benthamiana, leading to profound morphological changes and an 80% reduction in photosynthetic pigments [20]. Despite the impressive data that have been accumulated of the DXP pathway in plants and bacteria, knowledge about the regulatory mechanisms and the coordination with related pathways is still limited, as is knowledge about the cross-talk, that is, the exchange and transport of intermediates between the two isoprenoid pathways in plants. Moreover, the absence of the DXP pathway in mammals makes it an ideal target for antibiotics and herbicides. To this point, there is only one inhibitor, fosmidomycin, that has been reported to speciWcally block the DXP reductoisomerase and that is being investigated clinically [21]. In addition to its importance as a target for inhibitors, the DXP pathway may play a role in immune response and lead to the development of new immunomodulatory agents given that intermediates have been reported to be potent
 Tcell activators [22]. A primary requirement to address these issues is the development of adequate analytical procedures that provide separation and quantiWcation of the intermediates. Numerous procedures for the analysis of phosphorylated compounds have been described, but the majority of methods are based on either ion pair chromatography or anion exchange chromatography. The latter has been improved by the development of high-pH anion exchange methods in combination with conductometric detection [23]. With regard to the

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former, Kleinig and Beyer [24] were the Wrst to use the ion pair chromatography technique for the analysis of short-chain prenyl phosphates. This method was subsequently reWned and applied to the separation of all intermediates of the MVA pathway by McCaskill and Croteau [25]. A reversed-phase high-performance liquid chromatography (HPLC) procedure based on gradient elution with acetonitrile and NH4HCO3 was described for the separation of C5 to C35 diphosphates by Zhang and Poulter [26]. To this point, no separation of all intermediates of the DXP pathway has been reported. In this article, we describe a reversed-phase ion pair HPLC method that allows the analysis by sensitive radiodetection of the intermediates of the DXP pathway and its application to chromoplast in vitro systems. We also identify a way of diVerentiating IDP (9) and DMADP (10), thereby providing adequate analytical methods to investigate the role of IDI in isoprenoid biosynthesis.

Materials and methods Enzymes and reagents Pyruvic acid (Na+ salt) (1) was purchased from Sigma–Aldrich. [1-14C]Isopentenyl diphosphate (9) (58 mCi/mmol) and D-[5-3H]glucose (17 Ci/mmol) were purchased from Amersham Biosciences. [1,2,314 C3]Pyruvic acid (Na+ salt) (1) (155.5 mCi/mmol), [1,214 C2]1-deoxy-D-xylulose 5-phosphate (3) (103.7 mCi/ mmol), and [1,3,4-14C3]2C-methyl-D-erythritol 2,4-cyclodiphosphate (7) (155.5 mCi/mmol) were synthesized as described previously [27]. The synthesis of [1-3H]2Cmethyl-D-erythritol 4-phosphate (4) (751 mCi/mmol) is described in [28], and [4-3H](E)-4-hydroxy-3-methylbut2-enyl diphosphate (8) (1.56 Ci/mmol) was synthesized as described previously [29]. [1,3,4-14C3]4-Diphosphocytidyl-2C-methyl-D-erythritol (5) and [1,3,4-14C3]4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (6) were synthesized using recombinant CMS and CMK protein (kindly provided by Henriette Alpermann). The proteins were expressed in E. coli BL21 (DE3) (kindly provided by Jon Page) transformed with plasmids pCR T7/NT Topo (Invitrogen) containing ampliWcates of the ispD and ispE genes from E. coli. Recombinant IDP/ DMADP isomerase from Cannabis sativa (kindly provided by Henriette Alpermann and Jon Page) was used for the synthesis of [1-14C]DMADP. The idi gene was ampliWed by PCR using a cDNA library as template, cloned into the plasmid pHIS8-3 [30], and transformed into E. coli BL21 (DE3) for expression of the recombinant protein. [1,2-14C2]1-Deoxy-D-xylulose (3a) (103.7 mCi/mmol), [1-3H]2C-methyl-D-erythritol (4a) (751 mCi/mmol), and [4-3H](E)-2-methylbut-2-ene-1,4diol (8b) (1.56 Ci/mmol) were synthesized by alkaline phosphatase treatment of the corresponding phosphates.

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Tetra-n-butylammonium hydrogen sulfate (TBAS) was purchased from VWR International. Solvents for HPLC analysis were of HPLC grade and purchased from VWR International as well. All other reagents were of analytical grade and purchased from Acros Organics. Alkaline phosphatase from bovine intestinal mucosa and inorganic pyrophosphatase from baker’s yeast were obtained from Sigma–Aldrich. HPLC system The HPLC system used was a Merck–Hitachi LaChrom interfaced with a radiodetector, Ramona 2000 (Raytest), equipped with a solid scintillation glass Xow cell (45 m particle size, 200 l volume, US scintillator) for the measurement of radioactivity. The energy window was set at 0 to 160 keV, and the counting eYciencies for 3H and 14C were 10 and 90%, respectively. The background was approximately 180 cpm. The detector signal was recorded and integrated by a personal computer and a chromatography software program (Winnie 32, version 2.0). The separation of DXP pathway intermediates was accomplished by reversed-phase ion pair HPLC on a Luna C8 (2) (5 m) 250 £ 4-mm column with a guard column (4 £ 3 mm) of the same material (Phenomenex). Gradient elution The analyses were performed using a modiWed HPLC method described previously by McCaskill and Croteau [25]. DXP pathway intermediates were eluted by a binary solvent system consisting of 10 mM TBAS in water, pH 6.0 (solvent A), and 10 mM TBAS in 70% (v/v) methanol, pH 6.0 (solvent B), as eluents. The solvents were prepared by the dilution of a 200-mM stock solution of TBAS in water (pH adjusted to 6.0 with solid Na2HPO4, Wnal concentration 320 mM) and were Wltered (0.45 m) and degassed before use. The elution program consisted of an isocratic Xow of 100% solvent A for 20 min, followed by a linear gradient to 60% solvent B:40% solvent A over the subsequent 60 min with a Xow rate of 0.75 ml/min. The column was regenerated by a switch from 60% solvent B to 100% solvent A within 5 min and was washed for another 15 min with 100% solvent A. The total elution time was 100 min. Then 50- to 100-l aliquots of the aqueous fractions, obtained after incubation with chromoplasts, were injected onto the column. Isolation and incubation of chromoplasts from Capsicum annuum and Narcissus pseudonarcissus Chromoplasts from the pericarp of red bell pepper or the inner coronae of N. pseudonarcissus were isolated and incubated with radiolabeled substrates as described previously [31]. BrieXy, the reaction mixtures contained

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100 mM Hepes (pH 7.6), 2 mM MnCl2, 10 mM MgCl2, 5 mM NaF, 2 mM NADP+, 1 mM NADPH, 6 mM ATP, chromoplasts equivalent to 1–2 mg of protein, and either 0.1 to 0.2 Ci of 14C-labeled substrates or 2 Ci of 3Hlabeled and up to 10 nmol of unlabeled substrates in a total volume of 500 l. The assay mixtures were incubated at 30 °C. To terminate the reaction, assay mixtures were brieXy heated, put on ice, and subsequently extracted with ethyl acetate. Aliquots from both the ethyl acetate and aqueous fractions were analyzed by liquid scintillation counting (Rotiscint scintillation Xuid, LS 6500, Beckmann). The remaining aqueous fractions were immediately frozen in liquid nitrogen and lyophilized, and the residue was resuspended in 100 l of water prior to HPLC analysis. Ethyl acetate fractions were evaporated to dryness, dissolved in a deWned volume of ethyl acetate, and analyzed by thin-layer chromatography (TLC) as described previously [32]. Hydrolysis of IDP (9) and DMADP (10) to the corresponding phosphates and alcohols The aqueous phase of assay mixtures, after extraction with ethyl acetate, was lyophilized and resuspended in 100 l of water. For complete dephosphorylation, 0.5 U alkaline phosphatase was added, whereas for conversion to the corresponding phosphates, 1 U inorganic pyrophosphatase was added and the mixture was incubated for 60 min at 37 °C. To terminate the enzymatic conversion, the assay mixtures were heated to 70 °C for 5 min and then immediately placed on ice and analyzed by reversed-phase ion pair HPLC. Incubation of IDP (9) with IDP/DMADP isomerase from C. sativa Reaction mixtures contained 100 mM Hepes (pH 7.6), 20 mM MgCl2, 10 mM MnCl2, 2.5 mM dithiothreitol (DTT), 0.2 Ci [1-14C]IDP (9) (58 mCi/mmol), and 1.8 g IDP/DMADP isomerase in a total volume of 100 l and were incubated for 1 h at 30 °C. For termination of the enzymatic conversion, assay mixtures were heated brieXy and then immediately placed on ice.

reversed-phase ion pair HPLC. This technique is based on the formation of undissociated neutral ion complexes by the addition of complex-forming agents, such as tetrabutylammonium salts, to the mobile phase, and these agents can be separated on a reversed-phase column. The combination with a radiodetector based on solid scintillation counting provided sensitive detection of the pathway intermediates, which usually lack a chromophore, and allowed us to study the Xux of metabolites of the DXP pathway. The detection limit was in the range of 900 cpm, which corresponds to approximately 1000 dpm in the case of 14C-labeled compounds and 9000 dpm in the case of 3H-labeled compounds based on counting eYciencies of 90 and 10%, respectively. McCaskill and Croteau described a reversed-phase HPLC method [25] that allows the analysis of all MVA pathway intermediates using a ternary solvent system with TBAS as ion pair reagent. With this method, the separation of pyruvate (1), nucleoside diphosphates and IDP (9), DMADP (10), and the long-chain prenyl diphosphates geranyl, farnesyl, and geranylgeranyl diphosphate was possible. It built the basis for the development of an elution system to separate DXP pathway intermediates. Because we focused on the analysis of the early DXP pathway metabolites prior to IDP (9) and DMADP (10), which are more hydrophilic due to the presence of hydroxyl groups or nucleotide moieties, we assumed that they should elute earlier from a reversedphase column. This assumption led to the shortening of the elution time to 100 min, including regeneration of the HPLC column, compared with 150 min (the elution time without column regeneration) of the system described by McCaskill and Croteau [25], thereby making our method more applicable to routine analysis. Moreover, the analyses required only a binary solvent system instead of a ternary one. A further improvement was the use of a C8 HPLC column instead of a C18 one given that the C8 column proved to be more robust and less sensitive to the ion pair reagent present in the eluent. Further precautions to prolong the lifetime of the column included extensive wash and regeneration cycles and frequent changes of the guard column. These treatment conditions allowed up to 1000 HPLC runs before a signiWcant shift in the retention times was observed.

Results and discussion

Separation of standard compounds

HPLC method for the separation of DXP pathway intermediates

The elution program consisted of a binary solvent system with an isocratic Xow of 10 mM TBAS in water (solvent A) for 20 min followed by a linear gradient of increasing methanol concentration up to 42% (v/v) within 60 min, keeping the concentration of TBAS at 10 mM. The addition of the ion pair reagent, TBAS, transfers the charged phosphate groups into undissociated hydrophobic complexes that can be separated on a reversed-phase column. The separation of standard

DXP pathway intermediates represent charged molecules, mostly mono- and diphosphates but also nucleotide derivatives, which can be separated by either anion exchange chromatography or ion pair chromatography. Because we were also interested in separating the dephosphorylated metabolites, the method of choice was

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compounds of DXP pathway intermediates, except for DMADP (10), is shown in Fig. 2. The more hydrophilic early metabolites [e.g., DXP (3), MEP (4)] eluted within the Wrst 20 min of isocratic Xow, whereas elution of the more lipophilic compounds [e.g., cMEDP (7), HMBDP (8)] and nucleotide derivatives [e.g., CDP–ME (5), CDP– MEP (6)] required increasing concentrations of methanol in the mobile phase. DX (3a) and ME (4a), which are not true intermediates but rather products of hydrolysis, are not separated in this chromatogram, in contrast to their respective phosphates, DXP (3) and MEP (4). Separation of metabolites formed in chromoplasts In plants, the DXP pathway is localized in plastids. Hence, isolated chromoplasts either from fruits of C. annuum or from daVodil Xowers, which are very well characterized and have been used extensively to study carotenoid biosynthesis [33,34] and more recently to investigate the DXP pathway [28,31], were employed as model systems to test the suitability of the HPLC method. Isolated chromoplasts from N. pseudonarcissus and C. annuum were incubated with [1,3,4-14C3]cMEDP (7) for 1 or 4 h and subsequently extracted with ethyl acetate, and the remaining aqueous phase was analyzed by radio HPLC. Ethyl acetate was found to eVectively extract all carotenoids present in these chromoplasts, composed of - and
-carotene, phytoXuene, phytoene, and the more polar xanthophylls such as lutein in the case of N. pseudonarcissus and capsanthin and capsorubin in the case of C. annuum [33,35]. Extraction of DXP

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pathway intermediates either did not occur or was only minor—less than 5% of DX (3a) and approximately 2– 2.5% of ME (4a). To avoid target molecule destruction, the extraction was performed at a neutral pH (Hepes, pH 7.6) and the remaining aqueous phase was immediately frozen in liquid nitrogen and lyophilized. Determination of the radioactivity in the ethyl acetate extract by liquid scintillation counting (up to 30% incorporation of the applied radioactivity) and TLC analysis of carotenoids, as described previously [32], indicated that the DXP pathway to carotenoids was fully functional. Labeling with [1,3,4-14C3]cMEDP (7) aVorded high levels of HMBDP (8) and IDP (9) for chromoplasts from N. pseudonarcissus (Fig. 3A), whereas with Capsicum chromoplasts only trace amounts of IDP (9) were detectable in addition to a peak resulting from HMBDP (8) (Fig. 4A). The retention times of the radiolabeled intermediates isolated from chromoplasts were in good agreement with those obtained with standard substances (Fig. 2). Separation of IDP (9), DMADP (10), and their respective monophosphates and alcohols The separation of IDP (9) and DMADP (10) by chromatographic methods represents a speciWc analytical problem, however, in determining their relative rates of formation by IDP synthase and IDP/DMADP isomerase [17]. McCaskill and Croteau [25] published the clear separation of IDP (9) and DMADP (10) with their ion pair HPLC system in 1993. When we adapted their sys-

Fig. 2. Reversed-phase ion pair HPLC separation of standards. The numbered peaks are as follows: 3, 1-deoxy-D-xylulose 5-phosphate; 3a, 1-deoxyD-xylulose; 4, 2C-methyl-D-erythritol 4-phosphate; 4a, 2C-methyl-D-erythritol; 5, 4-diphosphocytidyl-2C-methyl-D-erythritol; 6, 4-diphosphocytidyl2C-methyl-D-erythritol 2-phosphate; 7, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; 8, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate; 9, isopentenyl diphosphate.

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Fig. 3. Reversed-phase ion pair HPLC separation of the aqueous phase of an enzyme assay with chromoplasts from N. pseudonarcissus that had been incubated with 0.15 Ci [1,3,4-14C3]cMEDP (7) (15 mCi/mmol) for 1 h (A) and subsequently treated with alkaline phosphatase (B). The numbered peaks are as follows: 7, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; 8, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate; 8b, (E)-2-methylbut-2-ene1,4-diol; 9, isopentenyl diphosphate; 9a, isopentenyl phosphate; 9b, isopentenyl alcohol; 10, dimethylallyl diphosphate; 10a, dimethylallyl phosphate; 10b, dimethylallyl alcohol.

Fig. 4. Reversed-phase ion pair HPLC separation of the aqueous phase of an enzyme assay with chromoplasts from C. annuum that had been incubated with 0.15 Ci [1,3,4-14C3]cMEDP (7) (15 mCi/mmol) for 4 h (A) and subsequently treated with alkaline phosphatase (B). The numbered peaks are as follows: 7, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; 8, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate; 8b, (E)-2-methylbut-2-ene-1,4diol; 9, isopentenyl diphosphate; 9b, isopentenyl alcohol; 10, dimethylallyl diphosphate; 10b, dimethylallyl alcohol.

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tem to the separation of DXP pathway intermediates, we also intended to separate IDP (9) and DMADP (10). Because labeled DMADP (10) is not commercially available, we tried to generate [1-14C]DMADP (10) from [114 C]IDP (9) by an IDP/DMADP isomerase puriWed from E. coli (a gift from A. Bacher, Technical University, Munich, Germany). On HPLC analysis, we observed the formation of a metabolite that eluted approximately 15 min earlier than IDP (9) [29], as was reported for DMADP (10) by McCaskill and Croteau [25]. This seemed to conWrm that DMADP (10) can be separated from IDP (9), and we expressed this view in our article [29]. However, when doubts arose recently regarding the feasibility of separating IDP (9) and DMADP (10) by

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this ion pair HPLC system [17], we reevaluated our results using an IDP/DMADP isomerase cloned from C. sativa. Indeed, as stated by Rohdich and colleagues [17], IDP (9) and DMADP (10) could not be separated but eluted with the same retention time of approximately 78 min (Fig. 5A). The supposed DMADP peak [25,29] with a retention time of approximately 63 min was identiWed as isopentenyl phosphate (IP, 9a) by comparing the retention time with a reference compound generated by treating [1-14C]IDP (9) with an inorganic pyrophosphatase from baker’s yeast obtained from Sigma–Aldrich and by mass spectroscopy [20]. Therefore, we concluded that the sample of IDP/DMADP isomerase provided by A. Bacher in 1999 was contaminated by an inorganic

Fig. 5. Reversed-phase ion pair HPLC separation of [1-14C]IDP after treatment with IDP/DMADP isomerase cloned from C. sativa (A) and subsequent incubation with inorganic pyrophosphatase (B) or alkaline phosphatase (C). The numbered peaks are as follows: 9, isopentenyl diphosphate; 9a, isopentenyl phosphate; 9b, isopentenyl alcohol; 10, dimethylallyl diphosphate; 10a, dimethylallyl phosphate; 10b, dimethylallyl alcohol.

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pyrophosphatase. Recent results relating to virusinduced gene silencing of DXP pathway enzymes in N. benthamiana [20] proved that this pyrophosphatase not only exists in E. coli and yeast but also is present in plastids, where it might play a role in metabolic inactivation of high, and possibly inhibitory, IDP levels. In addition, IP (9a) is not a dead end product but rather can reenter the isoprenoid pathway [32]. Although IDP (9) and DMADP (10) cannot be separated with this HPLC system, the corresponding monophosphates, generated from the diphosphates by treatment with pyrophosphatase, elute with slightly diVerent retention times (Fig. 5B). An even better separation of the isomers can be achieved at the level of the alcohols formed by treatment with alkaline phosphatase (Fig. 5C), thereby allowing the quantiWcation of IDP (9) and DMADP (10). When applying this knowledge to the enzyme assays with chromoplasts, we could demonstrate that in both cases, IDP (9) and DMADP (10) were formed; however, the relative ratio of IDP/DMADP was approximately 1:2–3 for daVodil chromoplasts (Fig. 3B), which is indicative of the action of an IDP/DMADP isomerase [36], whereas it was approximately 5:1 for chromoplasts of C. annuum (Fig. 4B). The two peaks of IP (9a) and DMAP (10a) formed in daVodil chromoplasts after application of radiolabeled cMEDP (7) (Fig. 3A) could be due to the activity of the previously mentioned pyrophosphatase given that the levels of IDP (9) and DMADP (10) are quite high in these chromoplasts. In conclusion, the HPLC method described in this article allows the separation of the intermediates of the nonmevalonate isoprenoid pathway except IDP (9) and DMADP (10). However, the corresponding phosphates and alcohols, which are easily accessible by pyrophosphatase or phosphatase treatment, can be separated with this HPLC system, which should be adaptable to a variety of plant- and bacteria-derived systems. Hence, it will allow further investigation of the DXP pathway, its regulation, and its relation to other pathways. Moreover, the chromoplasts of N. pseudonarcissus and C. annuum used in these experiments represent excellent systems to elucidate the limiting role of an IDP/DMADP isomerase in the DXP pathway of isoprenoid biosynthesis in plants.

Acknowledgments We thank Henriette Alpermann for the synthesis of [1,3,4-14C3]4-diphosphocytidyl-2C-methyl-D-erythritol (5) and [1,3,4-14C3]4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (6). We are grateful to Jon Page and Henriette Alpermann for providing IDP/DMADP isomerase from C. sativa. This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn.

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