Studies on the nonmevalonate pathway of terpene biosynthesis The role of 2 C -methyl-D-erythritol 2,4-cyclodiphosphate in plants Monika Fellermeier 1 , Maja Raschke 1 , Silvia Sagner 1 , Juraithip Wungsintaweekul 2 , Christoph A. Schuhr 2 , Stefan Hecht 2 , Klaus Kis 2 , Tanja Radykewicz 2 , Petra Adam 2 , Felix Rohdich 2 , Wolfgang Eisenreich 2 , Adelbert Bacher 2 , Duilio Arigoni 3 and Meinhart H. Zenk 1 1 Biozentrum-Pharmazie, Universita ¨ t Halle, Halle/Saale, Germany; 2 Lehrstuhl fu ¨ r Organische Chemie und Biochemie, Technische Universita ¨ tMu ¨ nchen, Garching, Germany; 3 Laboratorium fu ¨ r Organische Chemie, Eidgeno ¨ ssische Technische Hochschule Ho ¨ nggerberg, Zu ¨ rich, Switzerland 2C-Methyl-D-erythritol 2,4-cyclodiphosphate was recently shown to be formed from 2C-methyl- D-erythritol 4-phos- phate by the consecutive action of IspD, IspE, and IspF proteins in the nonmevalonate pathway of terpenoid biosynthesis. To complement previous work with radio- labelled precursors, we have now demonstrated that [U- 13 C 5 ]2C-methyl-D-erythritol 4-phosphate affords [U- 13 C 5 ]2C-methyl-D-erythritol 2,4-cyclodiphosphate in isolated chromoplasts of Capsicum annuum and Narcissus pseudonarcissus. Moreover, chromoplasts are shown to efficiently convert 2C-methyl- D-erythritol 4-phosphate as well as 2C-methyl- D-erythritol 2,4-cyclodiphosphate into the carotene precursor phytoene. The bulk of the kinetic data collected in competition experiments with radiolabeled substrates is consistent with the notion that the cyclodipho- sphate is an obligatory intermediate in the nonmevalonate pathway to terpenes. Studies with [2,2 0 - 13 C 2 ]2C-methyl- D-erythritol 2,4-cyclodiphosphate afforded phytoene characterized by pairs of jointly transferred 13 C atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundance, in positions 16/1, 4/5, 8/9, and 12/13. A detailed scheme is presented for correlating the observed partial scrambling of label with the known lack of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isomerase. Keywords: nonmevalonate pathway; terpene; chromoplasts; 2C-methyl- D-erythritol 2; 4-cyclodiphosphate. For a period of several decades, the mevalonate pathway elucidated in animal cells and yeast by the studies of Bloch, Cornforth and Lynen has been considered as the universal source of isoprenoid precursors for the biosynthesis of terpenoids (reviewed in [1–4]). In recent years, a second pathway was discovered in certain eubacteria and plants by the research groups of Rohmer and Arigoni (reviewed in [5– 7]). Specifically, the incorporation of 13 C-labeled acetate and glucose in bacteria such as Rhodopseudomonas palustris [8] and Escherichia coli [9], as well as in plants [10] suggested a triose and pyruvate as precursors for the formation of isoprenoids via the alternative pathway. Arigoni and his coworkers found that 1-deoxy- D-xylu- lose, a known precursor of the vitamins thiamine [11] and pyridoxol [12], could be incorporated into terpenoids by E. coli [9] as well as by higher plants [7]. More specifically, plants were shown to utilize the mevalonate pathway in the cytoplasmic compartment and the nonmevalonate pathway in the plastid compartment [7,10,13,14]. More recently, the origin of a variety of plant terpenoids could be assigned to this plastid-based nonmevalonate pathway (reviewed in [6]). Recent studies by several research groups identified 1-deoxy- D-xylulose 5-phosphate synthase as the first enzyme of the alternative terpenoid pathway in certain bacteria [15–17] and plants [18,19]. The enzyme product is converted into the branched chain polyol, 2C-methyl- D-erythritol 4-phosphate, by a reductoisomerase via a skeletal rearrangement followed by an NADPH-dependent reduction [20– 23]. We have shown that in E. coli 2C-methyl- D-erythritol 4-phosphate can be converted into a cyclic diphosphate by the consecutive action of 4-diphosphocytidyl-2C-methyl- D- erythritol synthase, 4-diphosphocytidyl-2C-methyl- D-erythritol kinase and 2C-methyl- D-erythritol 2,4-cyclodiphosphate synthase [24–26] (Fig. 1). In the meantime, some of these results have been confirmed by other authors [27–29]. We have also shown that 14 C-labelled 2C-methyl-D-ery- thritol 2,4-cyclodiphosphate is incorporated into the lipid fraction of Capsicum annuum chromoplasts [26]. 2C-methyl- D-erythritol 2,4-cyclodiphosphate had been isolated earlier as a stress metabolite from bacterial cultures in high yield [30,31]. In this paper we describe the kinetics of 2C-methyl- D-erythritol 2,4-cyclodiphosphate incorporations into chromoplast preparations of C. annuum and Narcissus pseudonarcissus, as well as the incorporation of [U- 13 C 5 ]2- C-methyl- D-erythritol 2,4-cyclodiphosphate into phytoene from chromoplasts of C. annuum. EXPERIMENTAL PROCEDURES Materials [1- 3 H]2C-methyl-D-erythritol 4-phosphate was prepared according to a method described by Kis et al. using sodium Correspondence to W. Eisenreich, Lehrstuhl fu ¨ r Organische Chemie und Biochemie, Technische Universita ¨ tMu ¨ nchen, Lichtenbergstr. 4, D-85747 Garching, Germany. Fax: þ 49 89 289 13363, Tel.: þ 49 89 289 13043, E-mail: wolfgang.eisenreich@ch.tum.de (Received 5 July 2001, revised 8 October 2001, accepted 9 October 2001) Eur. J. Biochem. 268, 6302–6310 (2001) q FEBS 2001 Fig. 1. Biosynthesis of phytoene via the nonmevalonate pathway. q FEBS 2001 Isoprenoid biosynthesis in plants (Eur. J. Biochem. 268) 6303 [ 3 H]borohydride as reducing agent [32] [2,2 0 - 13 C 2 ]-, [2- 14 C]2C-methyl-D-erythritol 2,4-cyclodiphosphate, and [U- 13 C 5 ]2C-methyl-D-erythritol 4-phosphate were prepared as described [33,34]. Isolation of chromoplasts from C. annuum Chromoplasts were isolated by a slight modification of the method first described by Camara [35,36]. Pericarp of red pepper (500 g) was homogenized at 4 8C in 600 mL of 50 m M Hepes, pH 8.0, containing 1 mM dithioerythritol, 1 mM EDTA and 0.4 M sucrose (buffer A). The suspension was filtered through four layers of nylon cloth (50 mm) and centrifuged (10 min, 3290 g, GSA rotor) to obtain a pellet of crude chromoplasts which was homogenized in 400 mL of buffer A. The suspension was centrifuged (10 min, 3290 g, GSA rotor). The pellet was homogenized and resuspended in 3 mL of 50 m M Hepes, pH 7.6, containing 1 mM 1,4- dithioerythritol. The suspension was filtered through one layer of nylon cloth (50 mm). Preparation of a chromoplast extract A suspension of washed C. annuum chromoplasts (5 mL; protein concentration, 10 –15 mg·mL 21 ) was diluted with 50 m M Hepes, pH 7.6, containing 1 mM dithioerythritol to a final volume of 40 mL. The mixture was kept for 10 min at 4 8C and was then centrifuged (60 min, 110 560 g,Ti50 rotor). The supernatant was applied to aSephadex G-25column (type PD-10, Amersham Pharmacia Biotech) which had been equilibrated with 50 m M Hepes, pH 7.6, containing 1 mM dithioerythritol. The column was developed with the same buffer. Fractions were combined and concentrated using a Centriprep-10 concentrator (Amicon). The final protein concentration was about 1– 2 mg·mL 21 . Isolation of chromoplasts from N. pseudonarcissus The isolation followed a procedure described by Kleinig & Beyer [37]. Inner coronae of N. pseudonarcissus (80 g) were homogenized in 250 mL of 67 m M Tris/HCl, pH 7.5, containing 5 m M MgCl 2 ,1mM dithioerythritol, 1 mM EDTA, 0.2% (w/v) polyvinylpyrrolidone K90, and 0.74 M sucrose. The suspension was filtered (three layers of nylon cloth, 50 mm) and centrifuged (5 min, 1990 g, GSA rotor). The supernatant was centrifuged (20 min, 25 400 g, GSA rotor) affording a pellet of crude chromoplasts which was resuspended in 2 mL of 67 m M Tris/HCl, pH 7.5, contain- ing 5 m M MgCl 2 ,1mM dithioerythritol and 50% (w/v) sucrose. The suspension was filtered through one layer of nylon cloth (50 mm). Aliquots of 2 mL were transferred to centrifuge tubes. Equal volumes of 40, 30 and 15% (w/v) sucrose in 67 m M Tris/HCl, pH 7.5, containing 5 mM MgCl 2 and 1 mM dithioerythritol were placed on top of the chromoplast suspension. Subsequent to centrifugation (60 min, 64 000 g, SW28 rotor), the fraction of intact chromoplasts at the 40/30% interphase was collected and diluted with 67 m M Tris/HCl containing 5 mM MgCl 2 and 1m M dithioerythritol to a final sucrose concentration of 15% (w/v). The suspension was centrifuged (20 min, 25 130 g, SS34 rotor) and the pellet was suspended in 2mLof67m M Tris/HCl, pH 7.5, containing 5 mM MgCl 2 and 1 mM dithioerythritol. Incorporation experiments with isotope-labeled substrates Reaction mixtures contained 100 m M Hepes, pH 7.6, 2 mM MnCl 2 ,10mM MgCl 2 ,5mM NaF, 2 mM NADP þ ,1mM NADPH, 6 mM ATP, 20 mM FAD and chromoplasts or chromoplast extract. Isotope-labeled 2C-methyl- D-erythritol 4-phosphate and/or 2C-methyl- D-erythritol 2,4-cyclodiphos- phate were added as indicated in Table 1. The mixtures were incubated at 30 8C. The reaction was terminated by ethyl acetate extraction. The lipid extract was dried over sodium sulfate. In experiments with radiolabeled substrates the residue was analyzed by scintillation counting and/or HPLC. The aqueous phase was analyzed by reversed phase ion pair HPLC monitored by scintillation counting. HPLC analysis of phosphorylated metabolites Reversed phase ion pair HPLC separations were performed with a Luna C8 column (Phenomenex, 5 mm, 4 £ 250 mm). The column was developed with a linear gradient of 0–42% methanol in 10 m M tetrabutylammo- nium sulfate, pH 6.0 (total volume, 60 mL; flow rate, 0.75 mL min 21 ). The retention volumes of 2C-methyl- D-erythritol 4-phosphate and 2C-methyl-D-erythritol 2,4-cyclodiphosphate were 10.0 and 29.0 mL, respectively. Table 1. Competition experiments with [1- 3 H]2C-methyl-D-erythritol 4-phosphate (MEP) and [2- 14 C]2C-methyl-D-erythritol 2,4-cyclodi- phosphate (cMEPP) in a chromoplast system from C. annuum. In each experiment, the sample volume was 150 mL. Conc., concentration; Sp. radioact., specific radioactivity. Proffered precursors Radioactivity incorporation into [2- 14 C]cMEPP [1- 3 H]MEP [1- 3 H]cMEPP the lipid-soluble material Experiment Conc. (m M) Sp. radioact. (mCi·mmol 21 ) Conc. (mM) Sp. radioact. (mCi·mmol 21 ) 14 C: 3 H (% a ) produced (nmol) 14 C (nmol) 3 H (nmol) 14 C: 3 H (% a ) A 0.040 20 0.01 360 80 : 20 0.1 2.2 0.3 88 : 12 B 0.040 20 0.08 45 33 : 67 0.9 2.0 2.3 46 : 54 C 0.040 20 0.68 4.40 5.5 : 94.5 6.8 1.8 8.5 17 : 83 D 0.040 20 6.68 0.41 0.6 : 99.4 13.0 0.4 8.0 4.7 : 95.3 a Relative molar contribution of 14 C vs. 3 H pool. 6304 M. Fellermeier et al. (Eur. J. Biochem. 268) q FEBS 2001 Isolation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate The reaction mixture was centrifuged and the supernatant was applied to a CHROMABONDw SB column (500 mg, Macherey & Nagel). The column was washed with water and developed with 0.1 M ammonium bicarbonate. The effluent was passed through a column of DOWEX AG 50 W-X8 (100 –200 mesh, H þ -form) and was then lyophilized. The residue was dissolved in water and applied to a Nucleosil 10SB HPLC-column which was developed with 100 m M ammonium formate in 40% (v/v) methanol at a flow rate of 1 mL·min 21 . The effluent was monitored by scintillation counting. The retention time of 2C-methyl- D-erythritol 2,4-cyclodiphosphate was 26 min Fractions were combined and lyophilized. Isolation of phytoene Fresh red peppers (107 g) were homogenized and lyophilized. The dry powder was extracted with ethyl acetate (2.5 L). The solution was brought to dryness under reduced pressure. The residue was dissolved in 10 mL of a hexane/ethylacetate mixture (1 : 1, v/v). The solution was placed on a column of silica gel 60 (5 £ 40 cm) which was developed with a mixture of a hexane/ethylacetate (1 : 1; v/v). The red-colored fraction (400 – 480 mL) was collected and concentrated under reduced pressure. The residue was dissolved in 100 mL of chloroform. Purification of phytoene Aliquots (10 mL) of crude phytoene solution in chloroform were applied to a Hypersil RP18 HPLC column (5 mm, 4.5 £ 250 mm, ThermoQuest Germna GmbH, Egelsbach, Germany) that was developed with a mixture of isopropanol/ acetonitrile/water (50 : 45 : 5, v/v). The effluent was monitored photometrically (280 nm). The retention volumes of b-carotene, phytoene, and xanthophylls were 12, 14, and 20 mL, respectively. NMR spectroscopy NMR spectra were recorded with a DRX 500 spectrometer from Bruker Instruments (Karlsruhe, Germany) equipped with four channels and a pulsed gradient unit. Two dimensional homocorrelation and heterocorrelation experi- ments were performed with XWINNMR software from Bruker Instruments. Phytoene was measured in CDCl 3 ,and 2C-methyl- D-erythritol 2,4-cyclodiphosphate was measured in D 2 O. RESULTS Isolated chromoplasts of C. annuum were incubated with mixtures of [1- 3 H]2C-methyl-D-erythritol 4-phosphate and [2- 14 C]2C-methyl-D-erythritol 2,4-cyclodiphosphate (Table 1) and were then extracted with ethyl acetate. The aqueous phase was analyzed by HPLC in order to monitor the conversion of [1- 3 H]2C-methyl-D-erythritol 4-phosphate into the corresponding 2,4-cyclodiphosphate. The organic phase was analyzed for 3 H and 14 C in order to monitor the transformation of the proffered radioactive compounds into lipid-soluble material. The data summarized in Table 1 indicate that [1- 3 H]2C-methyl-D-erythritol 4-phosphate was diverted effici- ently to the 2C-methyl- D-erythritol 2,4-cyclodiphosphate Fig. 3. 13 C NMR signals of 2C-methyl-D-erythritol 2,4-cyclodiphosphate obtained by incubation of [U- 13 C 5 ]2C-methyl-D-erythritol 4-phosphate with a chromoplast extract of C. annuum. 13 C and 31 P coupling patterns are indicated. Fig. 2. Diversion of radioactivity from [2- 14 C]2C-methyl-D-ery- thritol 2,4-cyclodiphosphate into lipid-soluble material of chromo- plasts from Narcissus pseudonarcissus. (A) 2C-methyl- D-erythritol 2,4-cyclodiphosphate; (B) lipid-soluble fraction. q FEBS 2001 Isoprenoid biosynthesis in plants (Eur. J. Biochem. 268) 6305 pool. The amount of newly formed [1- 3 H]2C-methyl- D-erythritol 2,4-cyclodiphosphate increased with the con- centration of the proffered [1- 3 H]2C-methyl-D-erythritol 4-phosphate; the transformation showed saturation characteristics. [1- 3 H]2C-methyl-D-erythritol 4-phosphate as well as [2- 14 C]2C-methyl-D-erythritol 2,4-cyclodiphosphate were efficiently converted into lipid-soluble material. The amount of [1- 3 H]2C-methyl-D-erythritol 4-phosphate converted into lipid-soluble material increased with the concentration of the profferred substrate; saturation was reached at a substrate concentration of less than 1 m M. The transformation of 14 C-labeled cyclic diphosphate into lipid-soluble compounds had its maximum efficacy (approximately 35%) at low concentrations of proffered [1- 3 H]2C-methyl-D-erythritol 4-phosphate. At high concen- trations of this compound, the incorporation of 14 C-label from the cyclic diphosphate into the lipid-soluble fraction was significantly diminished. In a similar experiment, we studied the formation of lipid- soluble material from [2- 14 C]2C-methyl-D-erythritol Fig. 4. 13 C NMR signals of phytoene obtained from [2,2 0 - 13 C 2 ]2- C-methyl- D-erythritol 2,4-cyclodiphosphate by incubation with chromoplasts of C. annuum. 13 C coupling patterns are indicated. Table 2. 13 C NMR assignments for phytoene. Position 13 C-Chemical shift (d, p.p.m.) a J CC b (Hz) INADEQUATE b 1, 1 0 131.23 42.3 17, 17 0 2, 2 0 123.97 3, 3 0 26.77 4, 4 0 39.72 42.5, 3.5 5, 5 0 d 134.93 42.5 18, 18 0 6, 6 0 124.22 7, 7 0 26.74 8, 8 0 39.72 42.5, 3.5 9, 9 0 d 135.33 42.2 19, 19 0 10, 10 0 124.41 11, 11 0 26.68 12, 12 0 40.49 40.7 13, 13 0 139.51 42.0 20, 20 0 14, 14 0 120.22 15, 15 0 123.35 16, 16 0 25.69 43.3 17, 17 0 17.68 42.2 18, 18 0 16.00 42.2 19, 19 0 16.04 42.2 20, 20 0 16.52 42.0 a Referenced to external TMS; b from the experiment with [2,2 0 - 13 C 2 ]2- C-methyl- D-erythritol 2,4-cyclodiphosphate; c–e assignments may be interchanged. Table 3. 13 C-Labeling pattern of phytoene obtained from chromo- plasts of C. annuum incubated with [2,2 0 - 13 C 2 ]2C-methyl- D-erythritol 2,4-cyclodiphosphate. ND, not determined, due to signal overlapping. Position 13 C a (%) 13 C– 13 C b (%) 1, 1 0 ND 86 2, 2 0 1.1 3, 3 0 c 1.1 4, 4 0 ND 31 5, 5 0 d ND 85 6, 6 0 1.3 7, 7 0 c 1.3 8, 8 0 ND 31 9, 9 0 d ND 85 10, 10 0 1.1 11, 11 0 c 1.2 12, 12 0 1.5 25 13, 13 0 10.5 86 14, 14 0 1.1 15, 15 0 1.0 16, 16 0 1.8 31 17, 17 0 9.0 82 18, 18 0 c 9.8 82 19, 19 0 c 8.5 83 20, 20 0 8.9 86 a Calculated as the relative 13 C abundance by comparison of 13 C NMR signal intensities of the labeled sample with 13 C NMR signal intensities of an unlabeled phytoene sample. b calculated as the fraction of the 13 C-coupled satellites in the global 13 C NMR intensity of a given atom. c–e assignments may be interchanged. 6306 M. Fellermeier et al. (Eur. J. Biochem. 268) q FEBS 2001 2,4-cyclodiphosphate using isolated chromoplasts from N. pseudonarcissus (Fig. 2). The incorporation of radio- actvity into lipid-soluble material was again checked by solvent extraction of reaction mixtures and the consumption of 2C-methyl- D-erythritol 2,4-cyclodiphosphate was monitored by HPLC analysis of the aqueous phase using a scintillation detector. As shown in Fig. 2, the radioactive substrate was virtually completely depleted and up to 94% of the proffered radioactivity was transformed into lipid- soluble material. Next, experiments with precursors labeled with stable isotopes were initiated. An extract prepared from isolated chromoplasts of C. annuum was depleted of low molecular mass compounds by gel filtration and was then incubated with [U- 13 C 5 ]2C-methyl-D-erythritol 4-phosphate in admix- ture of a small amount of [2- 14 C]2C-methyl-D-erythritol 4-phosphate at 30 8C for 15 h as described under Experimental procedures. A radioactive product was then isolated from the reaction mixture and was analyzed by 13 C NMR spectroscopy (Fig. 3). All 13 C NMR signals of the isolated compound were multiplets due to 13 C 13 C coupling. Based on chemical shift values and coupling constants, the compound was identified as 2C-methyl- D-erythritol 2,4-cyclodiphosphate (see [26] for NMR data of the authentic compound). The absence of singlet signals for the carbon atoms 1, 2, 2-Me, 3 and 4 in the spectrum of the isolated material demonstrates that the proffered material had not been diluted by significant amounts of endogenous material with natural 13 C abun- dance. It follows that the chromoplast extract used did not contain significant amounts of endogenous, unlabeled 2C-methyl- D-erythritol 2,4-cyclodiphosphate. Isolated chromoplasts from C. annuum were sub- sequently incubated with 0.7 m M [2,2 0 - 13 C 2 ]2C-methyl- D-erythritol 2,4-cyclodiphosphate at 30 8C for 12 h. The suspension was extracted with ethyl acetate, and phytoene (Fig. 1, compound 10) was isolated from the resulting mixture of lipophilic compounds. 13 C NMR signals of the isolated compound are shown in Fig. 4. Signal assignments taken from [38] are supported by 1- and 2- dimensional analysis of the 13 C-labeled phytoene sample (Table 2). Eight of the 20 13 C signals of the labeled phytoene appeared as singlets, eight signals showed high- intensity satellites caused by 13 C– 13 C coupling, and four signals were characterized by 13 C– 13 C coupling satellites of lower intensity (Table 3). The 13 C connectivity was further analyzed by a two-dimensional INADEQUATE experiment showing four pairs of 13 C atoms (Fig. 5). The terminal moiety of phytoene is biosynthetically derived from dimethylalkyl diphosphate. Both methyl groups (i.e. C-16 and C-17) of this moiety showed 13 C– 13 C coupling satellites, albeit of different intensities (Table 3). The labeling pattern of the reconstructed DMAPP unit is summarized in Fig. 6 and the evaluation of the signal intensities indicated a ratio of 10 : 1 for the two isotopomers a and b. The 13 C NMR signals of the methyl groups C-18, C-19, and C-20 of phytoene (biosynthetically equivalent to C-5 of IPP) showed 13 C-coupled satellites of high intensity (Table 3). From the signal intensities the molar fraction of the IPP isotopomer c can be calculated (Fig. 6). The signals of C-12 and the coincident signals of carbon atoms 4 and 8 showed one bond 13 C– 13 C coupling satellites of lower intensities that were substantially broadened by comparison with the central signal (Fig. 4). When processed for maximum resolution, these satellites appeared as pseudotriplets that could be due to long range coupling involving vicinal isoprenoid moieties. Due to the line broadening, the precision of signal integration is substan- tially reduced. However, within the experimental limits, it appears that the abundance of IPP isotopomer d is similar to that of DMAPP isotopomer b (Fig. 6). DISCUSSION The conversion of 2C-methyl-D-erythritol 4-phosphate into the corresponding 2,4-cyclodiphosphate by the consecutive action of three recombinant E. coli enzymes (specified by the ispD, ispE and ispF genes) has been described [24–29]. Orthologs of ispD and ispE from Arabidopsis thaliana and tomato, respectively, have been expressed in recombinant E. coli cells [39,40]. This paper shows that isolated chromoplasts from C. annuum and N. pseudonarcissus catalyze the con- version of 2C-methyl- D-erythritol 4-phosphate into the 2,4-cyclodiphosphate in a process that displays saturation kinetics and that the product of this reaction is further processed efficiently into phytoene. The results of the competition experiments summarized in Table 1 demonstrate that the incorporation of radioactivity from [1- 14 C]2C-methyl-D-erythritol 2,4-cyclodiphosphate Fig. 5. Two-dimensional INADEQUATE spectrum of phytoene obtained from [2,2 0 - 13 C 2 ]2C-methyl-D-erythritol 2,4-cyclodipho- sphate by incubation with chromoplasts of C. annuum. Fig. 6. Reconstruction of the labeling pattern of IPP (isotopomers a and b) and DMAPP (isotopomers c and d) from the labeling pattern of the phytoene sample obtained in the experiment with [2,2 0 - 13 C 2 ]2C-methyl-D-erythritol 2,4-cyclodiphosphate. Bold lines denote bonds linking adjacent 13 C atoms, numbers indicate the percentage molar fraction of the isotopomers. q FEBS 2001 Isoprenoid biosynthesis in plants (Eur. J. Biochem. 268) 6307 into phytoene (the main labeled component of the lipid- soluble fraction) is systematically diminished by the addition of increasing amounts of [1- 3 H]2C-methyl-D-ery- thritol 4-phosphate. Moreover, the data show that even at saturating concentrations of the tritiated compound, the relative transfer of 14 C-label from the cyclodiphosphate pool is always in excess of the value calculated from the original molar concentration of the two precursors. This requires that within the nonmevalonate pathway the cyclodiphosphate is nearer than the 4-phosphate to IPP and DMAPP, the two C5 building blocks from which phytoene is assembled. Thus, on all the available accounts the cyclodiphosphate behaves as expected for an obligatory intermediate. NMR spectroscopic analysis of the phytoene specimen obtained from [2,2 0 - 13 C 2 ]2C-methyl-D-erythritol 2,4-cyclo- diphosphate reveals a partial scrambling of label between (Z)- and (E)-methyl groups of DMAPP derived units and for the corresponding IPP-derived internal units. A similar partial scrambling of label between the (Z)- and (E)-methyls of the starter DMAPP unit matched by a corresponding scrambling within nonstarter C 5 -units derived from IPP in the elongation process has been observed in the mevalonate independent biosynthesis of carotenoids in cell cultures of Catharanthus roseus [13] as well as for the DMAPP-derived C 5 -unit of mevalonoid origin present in several clavine alkaloids [41–43]; but for a possible exception [44], a corresponding scrambling within the nonstarter C 5 units of mevalonoid terpenes seems to have gone undetected, probably because of the inadequacy of the analytical tools employed in earlier work using a 14 C label. In all the cases in which such a scrambling was observed it was usually ascribed to a lack of fidelity of the isomerase that inter- converts IPP and DMAPP. Participation of the isomerase is of crucial importance in the mevalonate pathway, in which formation of IPP and DMAPP take place in sequential steps; in contrast, the available evidence indicates that within the new pathway IPP and DMAPP are formed in independent steps from a common and yet unidentified intermediate [45–50], but a subsequent partial equilibration of the pre- formed units can nevertheless occur in organisms equipped with the isomerase, as is the case in higher plants in which the two metabolic pathways are known to coexist. The isomerization of IPP to DMAPP is an antarafacial process in which a proton is added to the re-re face of the double bond with subsequent or concomitant stereospecific removal of the H B hydrogen at C-2 (Fig. 7) from the opposite face of the molecule [51]; in the specific case of a recombinant yeast enzyme, the catalytic groups have been identified as Cys139, respectively, Glu207 [52]. In refinement and extension of previous observations by other authors [53], the Poulter group has carried out a thorough investigation on the lack of fidelity of this isomerase by analyzing the proton exchange that occurs when IPP is incubated with the enzyme in D 2 O [54]; a rapid exchange was detected for the C-4 hydrogens and one of C-2 hydrogens of IPP as well as for the (E)-methyl group of DMAPP, followed by a slower exchange (2% of the isomerization rate) of the methyl group of IPP and the (Z)-methyl group of DMAPP, and an even slower exchange (0.5% of the isomerization rate) of the olefinic hydrogen of DMAPP corresponding to the H A -hydrogen at C-2 of IPP. It is tempting to correlate this lack of regiochemical and stereochemical fidelity of the enzyme with the bidentate nature of the Glu207 carboxylate group positioned in the ES complex of the reaction as indicated in Fig. 7; in this geometric arrangement one of the oxygens is competent for efficient removal of the H B hydrogen from C-2 of the Fig. 7. Model representation for the positioning of the substrate in the active site of IPP isomerase; (a) and (b) represent alternative paths for proton abstraction from the substrate by the ambident carboxylate group of Glu207. Fig. 8. A detailed mechanistic scheme accounting for the known lack of fidelity of IPP isomerase. The resulting isotopic scrambling can be visualized by following the fate of the C atom labeled with a black dot in the starting material represented in the squares. The alternative reaction paths (a) and (b) correspond to the ones illustrated in Fig. 7. Sets A and B illustrate two different binding modes for the substrate. 6308 M. Fellermeier et al. (Eur. J. Biochem. 268) q FEBS 2001 substrate (path a), while the second oxygen is close enough to the methyl group to catalyze, as an alternative, the occasional removal of one of its hydrogens (path b). The outcome of the two competing deprotonation paths is illustrated in Fig. 8 for the predominant ES complex A of a sample of IPP carrying a 13 C label in its methyl group; a similar scheme involving a less stable ES complex B is necessary to account for the observed very slow exchange of the H A -hydrogen of IPP. In both cases, scrambling of the label takes place within the IPP pool and the error is then transcribed into the DMAPP pool by the normal action of the isomerase. The validity of the proposed scheme is rewardingly supported by the observation that the enzyme is capable to convert the IPP homolog X into its isomer Y (see Fig. 9) in a process which bypasses the formation of allylic isomers [53]. ACKNOWLEDGEMENTS This work was supported by grants from the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (SFB369) to A. B., W. E. and M. H. Z. and a fellowship from the Hans-Fischer- Gesellschaft to T. R. We thank Katrin Ga ¨ rtner for skillfull assistence and Prof B. Camara, Strasbourg, for a sample of phytoene. Financial support by Novartis International AG Basel (to D. A.) is gratefully acknowledged. REFERENCES 1. Qureshi, N. & Porter, J.W. (1981) Biosynthesis of mevalonic acid from acetyl-CoA. In Biosynthesis of Isoprenoid Compounds (Porter, J.W. & Spurgeon, S.L., eds), Vol. 1, pp. 47– 94. John Wiley, New York, USA. 2. Bloch, K. (1992) Sterol molecule: structure, biosynthesis and function. Steroids 57, 378–382. 3. Bach, T.J. 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