Tài liệu Báo cáo khoa học: Isoprenoid biosynthesis via the methylerythritol phosphate pathway Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase ppt

Eur J Biochem 269, 4446–4457 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03150.x Isoprenoid biosynthesis via the methylerythritol phosphate pathway Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase Jean-Francois Hoeffler, Denis Tritsch, Catherine Grosdemange-Billiard and Michel Rohmer ¸ Universite´ Louis Pasteur/CNRS, Institut Le Bel, Strasbourg, France The 1-deoxyxylulose 5-phosphate reductoisomerase (DXR, EC 1.1.1.267) catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP) This transformation is a two-step process involving a rearrangement of DXP into the putative intermediate 2-C-methyl-D-erythrose 4-phosphate followed by a NADPH-dependent reduction of the latter aldehyde By using [1-13C]DXP as a substrate, the rearrangement of DXP into [5-13C]2-C-methyl-D-erythrose 4-phosphate was shown to be NADPH dependent, although it does not involve a reduction step The putative aldehyde intermediate, obtained by chemical synthesis, was converted into MEP by the DXR in the presence of NADPH and into DXP in the presence of NADP+, indicating the reversibility of the reaction catalyzed by the DXR This reversibility was confirmed by the conversion of MEP into DXP in the presence of NADP+ The equilibrium was, however, largely displaced in favour of the formation of MEP The reduction step required the presence of a divalent cation such as Mg2+ or Mn2+ Many bacteria, the unicellular green algae and the chloroplasts from phototrophic organisms synthesize their isoprenoids via the mevalonate-independent 2-C-methylD-erythritol phosphate (MEP) pathway (Fig 1) [1–3] The initial step of this route is the formation of 1-deoxyD-xylulose 5-phosphate (DXP) by the condensation of (hydroxyethyl)thiamine resulting from pyruvate decarboxylation on glyceraldehyde 3-phosphate catalyzed by the thiamine diphosphate-dependent DXP synthase (DXS) [4–6] The second enzyme of this biosynthetic pathway, the DXP reductoisomerase (DXR), catalyzes the transformation of DXP into MEP in two steps DXR is a class B dehydrogenase [7,8] The corresponding gene has now been cloned from Escherichia coli [9], Zymomonas mobilis [10], Mentha x piperita [11], Arabidopsis thaliana [12], Synechocystis sp [7], Streptomyces coelicolor [13] and Pseudomonas aeruginosa [14] In the postulated mechanism of the reaction catalyzed by the DXR, DXP is first rearranged into 2-C-methyl-erythrose-4-phosphate [15], which is subsequently reduced by NADPH to yield MEP The latter aldehyde intermediate was, however, never characterized, neither directly, nor indirectly It is apparently not released from the enzyme active site during the catalysis [16,17] Three reactions are successively performed on the MEP framework, yielding three additional intermediates of the MEP pathway: conversion of MEP into 4-diphosphocytidyl-2-C-methyl-D-erythritol [18,19], phosphorylation of the C-2 hydroxyl group of yielding [20,21] and conversion of into 2-C-methyl-D-erythritol 2,4-cyclodiphosphate [22,23] The two last steps of the pathway were identified by a combination of genetic and biochemical methods An E coli strain engineered for the utilization of exogenous mevalonate accumulated tritiumlabelled 2-C-methyl-D-erythritol 2,4-cyclodiphosphate upon incubation of [1-3H]-2-C-methyl-D-erythritol and after disruption of the gcpE gene, suggesting that 2-C-methylD-erythritol 2,4-cyclodiphosphate is the substrate of the GcpE protein [24] Incubation of [3-14C]-2-C-methylD-erythritol 2,4-cyclodiphosphate with a crude cell-free system from an E coli strain overexpressing gcpE resulted in the formation of 4-hydroxy-3-methylbut-2-enyl diphosphate [25,26] Deletion of the lytB gene in a similarly engineered E coli strain, resulted in the accumulation of the same diol diphosphate [27] In addition, feeding with uniformly labelled [U-13C5]-1-deoxy-D-xylulose E coli strains overexpressing the gene of the xylulose kinase (responsible for the phosphorylation of free 1-deoxy-Dxylulose) as well as of all genes of the enzymes downstream of gcpE or lytB resulted in the accumulation of uniformly labelled 4-hydroxy-3-methylbut-2-enyl diphosphate or of isopentenyl diphosphate (IPP) 10 and dimethylallyl diphosphate 11, respectively [28,29] The nature of the cofactors required for the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into IPP 10 and dimethylallyl diphosphate 11 is still a matter of investigation (Fig 1) This paper focuses on the two intriguing consecutive steps catalyzed by the DXR from E coli Recently, ´ Correspondence to M Rohmer, Universite Louis Pasteur/CNRS, Institut Le Bel, rue Blaise Pascal, 67070 Strasbourg Cedex, France Fax: +33 90241345, E-mail: mirohmer@chimie.u-strasbg.fr Abbreviations: AHIR, acetohydroxy acid isomeroreductase; H2-NADPH, dihydro-NADPH; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; H-DXR, His-tagged DXR; IPP, isopentenyl diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate Enzymes: acetohydroxy acid isomeroreductase (EC 1.1.1.86), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267), 1-deoxy-D-xylulose 5-phosphate synthase (EC 4.1.3.7), NADP-dependent alcohol dehydrogenase (EC 1.1.1.2) (Received 12 June 2002, accepted 24 July 2002) Keywords: isoprenoid, 2-C-methyl-D-erythritol 4-phosphate, 1-deoxyxylulose 5-phosphate reductoisomerase, 2-C-methylD-erythrose4-phosphate Ó FEBS 2002 Deoxyxylulose phosphate reductoisomerase (Eur J Biochem 269) 4447 Fig 2-C-Methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis experiments performed using different combinations of substrate, inhibitor and NADPH have been reported [17] They suggested that NADPH binds before the normal substrate DXP or an inhibitor such as fosmidomycin and were consistent with an ordered mechanism DXR, like all enzymes of the MEP pathway, is a potential target for inhibitors acting as antibacterial or antiparasitic drugs or as herbicides Fosmidomycin, an inhibitor of the latter enzyme [30], has been shown to be active against the parasite responsible for malaria [31] Knowledge of the intimate mechanism of the reaction catalyzed by DXR is required for the design of such inhibitors The following questions were thus addressed: (1) What is the role of methylerythrose phosphate in the conversion of DXP into MEP? (2) Is the cofactor NADPH required for the sole isomerization of DXP into methylerythrose phosphate? (3) Is the reaction catalyzed by DXR reversible? (4) What kind of mechanism is involved in the rearrangement leading to the branched MEP carbon skeleton? An a-ketol rearrangement or a retroaldolization/aldolization would both afford the same reaction product MATERIALS AND METHODS General methods Unlabelled DXP was prepared either enzymatically [32], or chemically (Hoeffler et al., unpublished results) [1-13C] DXP and [2-13C]DXP were prepared enzymatically from glyceraldehyde phosphate and from [3-13C]pyruvate (Isotec, Miamisburg, OH, USA) or from [2-13C]pyruvate (Isotec, Saclay, France), respectively MEP was obtained either by chemical synthesis [33], or by enzymatic synthesis [9] [5-13C]MEP or [2-13C]MEP were prepared enzymatically from [1-13C]DXP or [2-13C]DXP, respectively [2-13C] Glycerol was purchased from Euriso-top (Saclay, France) Unless otherwise indicated, substrates, coenzymes and enzymes were from Sigma Dihydro-NADPH (H2-NADPH) was synthesized from NADPH by catalytic hydrogenation as previously described [34] The concentration of H2-NADPH was determined from the absorbance at 263 nm (e ẳ 18 500 M)1ặcm)1) All enzymatic DXR assays were recorded on an Uvikon 933 spectrophotometer (Kontron Instruments) by following the variation of the NADPH concentration Glycoaldehyde 2-phosphate 12 was synthesized from glycerol 3-phosphate by treatment with sodium metaperiodate and purified by anion exchange chromatography D-Erythrose 4-phosphate was purchased from Fluka All nonaqueous reactions were run in dry solvents under an argon atmosphere ÔDried and concentratedÕ refers to the removal of residual amounts of water with anhydrous Na2SO4 followed by evaporation of the solvent on a rotary evaporator Flash chromatography [35] (Merck silica gel, 40–63 lm) and TLC (Merck 1.05553) were performed using the same solvent system TLC plates were developed by heating up to 100 °C after spraying with an ethanol solution of p-anisaldehyde (2.5%), sulfuric acid (3.5%) and acetic acid (1.6%) or with an ethanol solution of phosphomolybdic acid reagent (10% w/v) NMR spectra were recorded on a 4448 J.-F Hoeffler et al (Eur J Biochem 269) Ó FEBS 2002 Bruker AC200 spectrometer at 200 MHz for 1H-NMR and 50 MHz for 13C-NMR, on a Bruker AC300 at 300 MHz for H-NMR, at 75 MHz for 13C-NMR and at 121.5 MHz for 31 P-NMR and also on Bruker ARX 500 at 500 MHz for 1H-NMR, 125 MHz for 13C-NMR and 202 MHz for 31 P-NMR 31P NMR spectra were calibrated against an external H3PO4 standard (d ¼ 0.00 p.p.m.) NMR experiments were carried out in CDCl3 or D2O using as internal standard CHCl3 (d ¼ 7.26 p.p.m.), DHO (d ¼ 4.56 p.p.m.) for 1H-NMR and 13CDCl3 (d ¼ 77.03 p.p.m.) for 13 C-NMR Negative mode electrospray mass spectrometry was performed on a Hewlett Packard 1100MS spectrometer using acetonitrile/water (1 : 1) as solvent GC-MS by chemical ionization was performed on a Finnigan-MAT TSQ 700 spectrometer with a 70 eV ionization energy using i-butane as gas (20 mL) at )5 °C, was added tert-butyldimethylsilyl triflate (3.6 mL, 15.5 mmol, 1.5 equivalents) After h at )5 °C, the reaction was quenched with water (15 mL), diluted with diethyl ether (45 mL), washed with M HCl (15 mL), 5% aqueous NaHCO3 (15 mL) and brine (15 mL) The organic layer was dried and concentrated The residue was purified by flash chromatography to afford 16 as colourless oil (2.2 g, 97%, Rf ¼ 0.48, hexane/ethyl acetate, 80 : 20) 1HNMR (300 MHz, CDCl3): d ¼ 0.14 (3H, s); 0.16 (3H, s); 0.90 (9H, s); 2.21 (1H, dddd, J ¼ 12.6 Hz, J ¼ 9.1 Hz, J ¼ 9.1 Hz, J ¼ 8.6 Hz, 3-Ha); 2.46 (1H, dddd, J ¼ 12.6 Hz, J ¼ 9.1 Hz, J ¼ 6.4 Hz, J ¼ 3.4 Hz, 3-Hb); 4.18 (1H, ddd, J ¼ 9.1 Hz, J ¼ 9.1 Hz, J ¼ 6.4 Hz; 4-Ha); 4.38 (2H, m, 4-Hb and 2-H) 13C-NMR (75 MHz, CDCl3): d ¼ )4.90 (CH3); 17.86 (quaternary C); 25.57 (CH3); 38.12 (C-3); 68.08 (C-2); 76.08 (C-4); 175.63 (C-1) Synthesis of 2-C-methyl-D-erythrose 4-phosphate Synthesis of (3S )-3-hydroxypentan-2-one 5-phosphate 14 (3S)-3-(tert-Butyldimethylsiloxy)pentan-2-one 5-dibenzylphosphate 17 Methyl lithium (1.6 M solution in diethyl ether, 5.9 mL, 9.5 mmol, 1.1 equivalents) was added dropwise to a stirred solution cooled to )78 °C of silyl ether 16 (1.9 g, 8.5 mmol, equivalent) in tetrahydofuran (40 mL) After stirring at )78 °C for h, the reaction was quenched by addition of water (20 mL) and diluted with diethyl ether (40 mL) The organic layer was separated, and the aqueous layer was extracted with diethyl ether (3 · 40 mL) The combined organic extracts were dried and concentrated in vacuo to give crude (3S)-3-(tert-butyldimethylsiloxy)-2-methyltetrahydrofuran-2-ol (1.8 g, 90%) as a colourless oil corresponding to the mixture of diastereomers at C-2, which was used for the next step without further purification Dibenzylphosphorochloridate [37] (1.6 g, 5.4 mmol, 1.5 equivalents) was added under stirring to a solution of (3S)-3-(tert-butyldimethylsiloxy)-2-methyltetrahydrofuran-2-ol (1.0 g, 4.3 mmol, equivalent) in pyridine (20 mL) at °C The reaction was stirred at room temperature for h After quenching by addition of water (2 mL), the solvents were removed under vacuum by azeotropic distillation with toluene Flash chromatography gave 17 as colourless oil (340 mg, 16%, Rf ¼ 0.35, hexane/ethyl acetate, 60 : 40) 1H-NMR (300 MHz, CDCl3): d ¼ 0.03 (3H, s); 0.05 (3H, s); 0.90 (9H, s); 1.91 (1H, m, 4-Ha); 1.92 (1H, m, 4-Hb); 2.13 (3H, s, 1-H); 4.12 (3H, m, 3-H, 5-H); 5.02 (4H, m, CH2Ph); 7.28–7.35 (10H, m) 13C-NMR (75 MHz, CDCl3): d ¼ )5.18 (CH3); )4.99 (CH3); 18.03 (quaternary C); 25.30 (C-1); 25.66 (3 · CH3); 35.04 (d, J ¼ 6.6 Hz, C-4); 63.41 (d, J ¼ 4.9 Hz, C-5); 69.28 (d, J ¼ 4.9 Hz, · CH2); 75.06 (C-3); 127.94, 128.53, 135.74 and 135.90 (aromatic C); 210.97 (C-2) 31P-NMR (121.5 MHz, CDCl3): d ¼ )3.46 (S)-2-Hydroxy-c-butyrolactone tert-butyldimethylsilylether 16 To a solution of (S)-2-hydroxy-c-butyrolactone 15 (1.1 g, 10.3 mmol, equivalent) and 2,6-lutidine (3.0 mL, 25.8 mmol, 2.5 equivalents) in dichloromethane (3S)-3-Hydroxypentan-2-one 5-dibenzylphosphate 18 To a stirred solution of the silyl ether 17 (200 mg, 0.41 mmol, equivalent) in tetrahydofuran (5 mL) was added tetrabutylammonium fluoride (160 mg, 0.49 mmol, 1.2 equivalents) 2-O-Benzyl-2-C-methyl-D-erythrose 4-dibenzylphosphate 13 [33] was hydrogenated (200 mg, 0.41 mmol) over 10% Pd/C (20 mg) in methanol (10 mL) for 30 at room temperature and atmospheric pressure (Fig 2) The mixture was filtered, and the filtrate diluted in water (15 mL), concentrated in order to remove the methanol and treated with a M NaOH solution to reach pH 7.5, yielding a mixture of 2-C-methyl-D-erythrose 4-phosphate and its dimethylacetal This mixture was treated with an ion exchange resin (Dowex 50 W-X4, H+ form) in water (10 mL) at 37 °C for h [36] After filtration, the pH of the filtrate was adjusted to 7.5 with a M NaOH solution For NMR and mass spectra analyses, an aliquot of the solution was lyophilized to dryness to afford the sodium salt of 2-C-methyl-D-erythrose 4-phosphate This aldehyde cannot be stored pure or in concentrated solution and was accordingly kept in water solution The aldehyde concentration was determined using the DXR assay 1H-NMR (500 MHz, D2O, : mixture of the aldehyde and of the hydrate): d ¼ 0.93 (2H, s, CH3, hydrate); 1.10 (1H, s, CH3, aldehyde); 3.62 (2H, m, · 4-H); 3.76 (1H, m, 3-H); 4.74 (0.7H, s, 1-H, hydrate); 9.41 (0.3H, s, 1-H, aldehyde) 13CNMR (125 MHz, D2O): d ¼ 16.34 (CH3); 18.25 (CH3); 63.65 (d, J ¼ 4.8 Hz, C-4); 65.12 (d, J ¼ 4.8 Hz, C-4); 73.78 (d, J ¼ 7.2 Hz, C-3); 74.11 (d, J ¼ 7.2 Hz, C-3); 75.27 (C-2); 79.83 (C-2); 91.78 (C-1, hydrate); 205.72 (C-1, aldehyde) 31P-NMR (202 MHz, D2O): d ¼ 5.2 and 5.0 Electrospray MS: m/z ¼ 213 (M-H, molecular ion of the 2-C-methyl-D-erythrose 4-phosphate mono-anion) Fig Synthesis of 2-C-methyl-D-erythrose 4-phosphate (i) H2, Pd/C in methanol; (ii) DOWEX 50 W-X4Ò, H+, in water at 40 °C Ó FEBS 2002 Deoxyxylulose phosphate reductoisomerase (Eur J Biochem 269) 4449 The mixture was stirred at room temperature for 30 and evaporated to dryness, and the residue was purified by flash chromatography to afford 18 as a colourless oil (135 mg, 88%, Rf ¼ 0.41, ethyl acetate/hexane, 90 : 10) H-NMR (300 MHz, CDCl3): d ¼ 1.77 (1H, m, 4-Ha); 2.14 (1H, m, 4-Hb); 2.15 (3H, s, 1-H); 3.74 (1H, s, OH); 4.17 (3H, m, 3-H, 5-H); 4.97 (1H, d, J ¼ 11.8 Hz, CH2Ph), 5.01 (1H, d, J ¼ 11.6 Hz, CH2Ph), 5.04 (1H, d, J ¼ 11.8 Hz, CH2Ph), 5.08 (1H, d, J ¼ 11.6 Hz, CH2Ph); 7.34 (10H, m) 13C-NMR (75 MHz, CDCl3): d ¼ 25.11 (C-1); 33.79 (d, J ¼ 6.6 Hz, C-4); 63.56 (d, J ¼ 6.6 Hz, C-5); 69.38 (d, J ¼ 4.9 Hz, · CH2); 73.20 (C-3); 127.97, 128.56, 135.64 and 135.77 (aromatic C); 209.40 (C-2) 31P-NMR (121.5 MHz, CDCl3): d ¼ )3.35 (3S)-3-Hydroxypentan-2-one 5-phosphate 14 (3S)-3Hydroxypentan-2-one 5-dibenzylphosphate 18 (35 mg, 0.01 mmol) was hydrogenated over 10% Pd/C (4 mg) in ethanol (2 mL) for h at room temperature and atmospheric pressure The mixture was filtered, and the filtrate concentrated The residue was dissolved in water (1 mL), and the pH adjusted to 7.5 with a M NaOH solution The mixture was lyophilized to give the sodium salt of 18 (20 mg, 98%) 1H-NMR (200 MHz, D2O): d ¼ 1.60 (1H, m, 4-Ha); 1.93 (1H, m, 4-Hb); 2.01 (3H, s, 1-H); 3.69 (2H, ddd, J4a,5 ¼ J4b,5 ¼ J5,P ¼ 6.2 Hz, 5-H); 4.23 (1H, dd, J ¼ 8.9 Hz, J ¼ 3.7 Hz, 3-H) 13C-NMR (75 MHz, D2O): d ¼ 25.30 (C-1); 33.45 (C-4); 60.53 (C-5); 73.95 (C-3); 215.15 (C-2) 31P-NMR (121.5 MHz, D2O): d ¼ 4.25 Synthesis of (4S)-4-hydroxypentan-2-one 5-phosphate 19 (R)-(tert-Butyldiphenylsiloxymethyl)oxirane 21 To a stirred solution of (R)-glycidol 20 (2.7 g, 36.0 mmol, equivalent) and imidazole (3.0 g, 44 mmol, 1.2 equivalents) in dry dichloromethane (30 mL) was added at °C tertbutylchlorodiphenylsilane (10.5 mL, 40 mmol, 1.1 equivalents) After h at room temperature, the reaction mixture was poured into water (30 mL), and the organic layer was separated The aqueous layer was extracted three times with dichloromethane (250 mL) The combined extracts were dried, filtered, concentrated and purified by flash chromatography to give 21 as a colourless oil (10.4 g, 92%, Rf ¼ 0.21, hexane/ethyl acetate, 95 : 5) 1H-NMR (200 MHz, CDCl3): d ¼ 1.09 (9H, s); 2.63 (1H, dd, J ¼ 5.2 Hz, J ¼ 2.5 Hz, 3-Ha); 2.76 (1H, dd, J ¼ 5.2 Hz, J ¼ 4.2 Hz, 3-Hb); 3.15 (1H, m, 2-H); 3.73 (1H, dd, J ¼ 11.8 Hz, J ¼ 4.7 Hz, 1-Ha); 3.88 (1H, dd, J ¼ 11.8 Hz, J ¼ 3.2 Hz, 1-Hb); 7.36–7.49 (6H, m); 7.69– 7.75 (4H, m) 13C-NMR (50 MHz, CDCl3): d ¼ 19.27 (quaternary C); 26.78 (3 · CH3); 44.45 (C-3); 52.25 (C-2); 64.34 (C-1); 127.74, 129.77, 133.31 and 135.64 (aromatic C) (2S)-4-Methylpent-4-ene-1,2-diol 22 Into a flask equipped with a mechanical stirrer, an addition funnel and containing anhydrous copper iodide (110 mg, 0.58 mmol, 0.1 equivalents) was added tetrahydofuran (20 mL) After cooling at )30 °C, isoproprenylmagnesium bromide (0.5 M in tetrahydofuran, 58 mL, 28.8 mmol, equivalents) was drop wise added The temperature never exceeded )30 °C After stirring for 30 at )30 °C (R)-(tert-butyldiphenylsiloxymethyl)oxirane 21 (1.8 g, 5.8 mmol, equivalent) in tetrahydofuran (10 mL) was slowly added, maintaining the temperature at )30 °C After stirring for h at )30 °C, the reaction was quenched by addition of a saturated NH4Cl solution and warmed up to room temperature The reaction was filtered through a sintered glass funnel containing celite and the tetrahydofuran was removed under reduce pressure The filtrate was diluted with diethyl ether (50 mL) and the organic layer was washed with water (20 mL) and brine (20 mL), dried and concentrated in vacuo Purification by flash chromatography afforded (2S)-1-tert-butyldiphenylsiloxy-2-hydroxypent-4-ene as a colourless oil (1.9 g, 98%, Rf ¼ 0.28, ethyl acetate/hexane, 10 : 90) To a stirred solution of the former silyl ether (1.9 g, 5.7 mmol, equivalent) in tetrahydofuran (50 mL) was added tetrabutylammonium fluoride (2.0 g, 6.2 mmol, 1.1 equivalents) The mixture was stirred at room temperature for h and evaporated to dryness The residue was purified by flash chromatography to afford 22 as colourless oil (610 mg, 91%, Rf ¼ 0.33, ethyl acetate) 1H-NMR (200 MHz, CDCl3): d ¼ 1.74 (3H, s, 4-CH3); 2.14 (2H, m, · 3-H); 3.17 (2H, -OH); 3.42 (1H, dd, J ¼ 11.3 Hz, J ¼ 7.1 Hz, 1-Ha); 3.63 (1H, dd, J ¼ 11.3 Hz, J ¼ 3.0 Hz, 1-Hb); 3.84 (1H, m); 4.77 (1H, m, 2-H); 4.83 (1H, m, 4-H) 13 C NMR (50 MHz, CDCl3): d ¼ 22.42 (4-CH3); 41.69 (C-3); 66.44 (C-1); 69.69 (C-2); 113.35 (C-3); 141.93 (quaternary C-4) (2S)-2-Hydroxypent-4-en-1-ol dibenzylphosphate 23 Dibenzylphosphorochloridate [36] (1.6 g, 5.4 mmol, 1.2 equivalents) was added under stirring to a solution of (2S)4-methylpent-4-ene-1,2-diol 22 (520 mg, 4.5 mmol, equivalent) in pyridine (10 mL) at )40 °C during a period of 20 The reaction was stirred at )40 °C for h, quenched by addition of water (2 mL), and the solvents were removed under vacuum by azeotropic distillation with toluene Flash chromatography gave 23 as a colourless oil (920 mg, 54%) (Rf ¼ 0.24, ethyl acetate/hexane, 65 : 35) 1H-NMR (200 MHz, CDCl3): d ¼ 1.72 (3H, s, 4-CH3); 2.13 (2H, m, 3-H); 2.69 (1H, -OH); 3.93 (3H, m, 1-H, 2-H); 4.75 (1H, m, 5-Ha); 4.83 (1H, m, 5-Hb); 5.07 (4H, m, · -CH2Ph); 7.35 (10H, m) 13C-NMR (50 MHz, CDCl3): d ¼ 22.39 (4-CH3); 41.19 (C-3); 68.09 (d, J ¼ 6.1 Hz, C-2); 69.50 (d, J ¼ 5.1 Hz, · CH2); 71.49 (d, J ¼ 5.8 Hz, C-1); 113.64 (C-5); 127.97, 128.58, 135.60 and 135.74 (aromatic C); 141.33 (C-4) 31P-NMR (121.5 MHz, CDCl3): d ¼ )2.55 (4S)-4-Hydroxypentan-2-one 5-dibenzylphosphate 24 To a biphasic solution of (2S)-2-hydroxypent-4-en-1-ol dibenzylphosphate 23 (110 mg, 0.29 mmol, equivalent) and NaIO4 (263 mg, 1.2 mmol, 4.2 equivalents) in a mixture of acetonitrile/carbon tertrachloride/H2O (2 : : 3, mL) was added ruthenium trichloride (7 mg, 0.03 mmol, 0.1 equivalents) [38] After vigorous stirring for 15 at room temperature, water (10 mL) and dichloromethane (10 mL) were added, and the two phases were separated The upper aqueous phase was extracted four times with dichloromethane (4 · 25 mL) The combined organic extracts were dried and concentrated The residue was purified by flash chromatography to afford 24 as a colourless oil (88 mg, 80%, Rf ¼ 0.41, ethyl acetate/hexane, 80 : 20) 1H-NMR (200 MHz, CDCl3): d ¼ 2.13 (3H, s, 1-H); 2.55 (2H, m, 3-H); 3.40 (1H, broad s, -OH); 3.94 (2H, m, 5-H); 4.18 (1H, m, 4-H); 4.99 (1H, d, J ¼ 11.6 Hz, CH2Ph), 5.03 (1H, d, J ¼ 11.8 Hz, CH2Ph), 5.05 (1H, d, J ¼ 11.6 Hz, CH2Ph), 4450 J.-F Hoeffler et al (Eur J Biochem 269) 5.09 (1H, d, J ¼ 11.8 Hz, CH2Ph); 7.34 (10H, m); 13CNMR (50 MHz, CDCl3): d ¼ 30.70 (C-1); 45.73 (C-3); 66.45 (d, J ¼ 6.5 Hz, C-4); 69.55 (d, J ¼ 5.0 Hz, · CH2); 70.41 (d, J ¼ 6.2 Hz, C-5); 127.84, 128.01, 128.61, 135.57 and 135.68 (aromatic C); 207.82 (C-2) 31P-NMR (121.5 MHz, CDCl3): d ¼ )2.73 (4S)-4-Hydroxypentan-2-one 5-phosphate 19 (4S)-4Hydroxypentan-2-one 5-dibenzylphosphate 24 (55 mg, 0.15 mmol) was hydrogenated over 10% Pd/C (6 mg) in ethanol (2 mL) for h at room temperature and atmospheric pressure The mixture was filtered, and the filtrate and evaporated to dryness The residue was dissolved in water (1 mL), and the pH adjusted to 7.5 with a M NaOH solution The mixture was lyophilized to give the sodium salt of 19 (35 mg, 99%) 1H-NMR (500 MHz, D2O): d ¼ 2.06 (3H, s); 2.55 (1H, dd, J ¼ 16.8 Hz, J ¼ 8.9 Hz, 3-Ha); 2.61 (1H, dd, J ¼ 16.8 Hz, J ¼ 4.1 Hz, 3-Hb); 3.51 (1H, m, 5-Ha); 3.57 (1H, m, 5-Hb); 4.07 (1H, m, 4-H) 13 C-NMR (75 MHz, D2O): d ¼ 29.66 (CH3); 45.64 (CH2); 46.02 (d, J ¼ 19.4 Hz, CH2); 66.95 (d, J ¼ 12.1 Hz, CH); 213.59 (CO) 31P-NMR (121.5 MHz, D2O): d ¼ 4.10 Purification of His-tagged deoxyxylulose 5-phosphate reductoisomerase The coding region for the dxr gene from E coli was cloned into the pRSET vector (Invitrogen) between the BglII and HindIII restriction sites This vector contains a DNA sequence encoding for six histidine residues Plasmid pRSET-DXR was introduced into E coli strain BL21(DE3)pLysE After induction of enzyme expression by addition of IPTG (0.4 mM) at mid-log phase (OD600, 0.7) at 37 °C, the culture was incubated for additional h at the same temperature Cells (from · 500 mL cultures) were harvested by centrifugation and washed with water They were resuspended in a 50 mM Tris/HCl, 250 mM NaCl, mM 2-mercaptoethanol pH buffer (10 mL) and disrupted by sonication (8 · 30 s pulses at 40-W output, duty cycle 50%) with cooling in an ice bath The cell-free system was centrifuged at 18 000 g for 30 at °C in a Sigma 3K30 centrifuge The crude cell extract was applied on a column of Ni-nitrilotriacetic acid agarose (Qiagen, 0.8 · cm) equilibrated with the same buffer The column was first washed with the same buffer, and then with the buffer containing imidazole (5 mM) The enzyme was eluted by applying a linear gradient of imidazole (5–120 mM) in the same buffer (2 · 30 mL) Fractions containing His-tagged DXR (H-DXR) were pooled and concentrated by ultrafiltration on a Centricon 30 unit (Millipore) The enzymatic solution was dialysed against 50 mM Tris/HCl, 100 mM NaCl, dithiothreitol (2 mM), pH buffer by several concentration/dilution steps using Centricon 30 units The concentration of protein was determined using the method of Bradford [39] H-DXR enzymatic activity The enzymatic activity was determined routinely at 37 °C in a 50 mM Tris/HCl, mM MnCl2, mM dithiothreitol pH 7.5 buffer containing 0.15 mM NADPH and 0.5 mM DXP H-DXR was added to have an absorbance decrease of about 0.1 min)1 The rate was measured by following the Ó FEBS 2002 decrease of the absorbance at 340 nm due to the formation of NADP+ from NADPH To compare the kinetic parameters (Km and V) of DXP and 2-C-methyl-D-erythrose 4-phosphate 4, assays were carried out in a 50 mM triethanolamine/HCl, mM MnCl2 (or mM MgCl2), mM dithiothreitol, pH 7.7, at a fixed concentration of NADPH (0.15 mM) The concentration of DXP varied from 31 to 310 lM, while the concentration of 2-C-methyl-D-erythrose 4-phosphate varied from 93 to 620 lM The concentrations of the stock solutions of substrate were determined enzymatically using the H-DXR The enzyme (4.3 lg) was added lastly in order to initiate the reaction D-Erythrose 4-phosphate was tested as the substrate of H-DXR at concentrations up to mM and H-DXR concentrations up to 13 lgỈmL)1 The influence of D-erythrose 4-phosphate (1 mM) on the activity of H-DXR was checked with DXP (96 lM) as the substrate The kinetic parameters (Km and V) in the reverse reaction were determined at a fixed concentration of NADP+ (0.15 mM) The assays were performed at 37 °C in a 50 mM Tris/HCl, mM MnCl2, mM dithiothreitol pH 7.5 buffer The concentration of MEP varied from 75 to 375 lM The concentration of the stock solution of MEP was determined by titration of the phosphate according to the method of Leloir & Cardini [40] The enzyme (4 lg) was added lastly in order to initiate the reaction Reduction of 2-C-methyl-D-erythrose 4-phosphate to 2-C-methyl-D-erythritol 4-phosphate by H-DXR To show that the reduction of 2-C-methyl-D-erythrose 4-phosphate really gives 2-C-methyl-D-erythritol 4-phosphate 5, the aldehyde (10 mg) was treated overnight with DXR (1.2 mg) in the presence of NADPH (0.5 mM) in a triethanolamine/HCl, mM MnCl2, mM dithiothreitol pH 7.7 buffer at 37 °C (4 mL final volume) NADPH was regenerated using the isopropanol/alcohol dehydrogenase system from Thermoanaerobium brockii [41] After hydrolysis of the phosphate esters with alkaline phosphatase (bovine intestinal mucosa, Sigma, 0.5 mg) for h at 37 °C, the medium was lyophilized, and the residue was acetylated overnight with a mixture of acetic anhydride and pyridine (0.2 mL, : v/v) After evaporation of the reagents, the residue was analysed by TLC Methylerythritol triacetate was isolated and identified by 1H-NMR (Rf ¼ 0.41, ethyl acetate/hexane, 50 : 50) 1H NMR (200 MHz, CDCl3): d ¼ 1.24 (3H, s, CH3); 2.04 (3H, s, CH3COO); 2.09 (3H, s, CH3COO); 2.11 (3H, s, CH3COO); 2.49 (1H, s, OH); 3.89 (1H, d, J1a,1b ¼ 11.6 Hz, Ha); 4.15 (1H, d, J1a,1b ¼ 11.6 Hz, 1-Hb); 4.16 (1H, dd, J4a,4b ¼ 12.1 Hz, J3,4a ¼ 8.1 Hz, 4-Ha); 4.56 (1H, dd, J4a,4b ¼ 12.1 Hz, J3,4b ¼ 2.7 Hz, 4-Hb); 5.18 (1H, dd, J3,4a ¼ 8.1 Hz, J3,4b ¼ 2.7 Hz, 3-H); 13C-NMR (50 MHz, CDCl3): d ¼ 19.80 (CH3); 20.62 (CH3); 20.71 (CH3); 62.67 (CH2); 68.02 (CH2); 71.95 (quaternary C, C-2); 72.54 (CH, C-3); 169.99 (CO); 170.85 (2 · CO) Isomerization of 2-C-methyl-D-erythrose 4-phosphate into DXP by H-DXR 2-C-Methyl-D-erythrose 4-phosphate (10 mg) was treated overnight with H-DXR (1.2 mg) in the presence of NADP+ Ó FEBS 2002 Deoxyxylulose phosphate reductoisomerase (Eur J Biochem 269) 4451 (0.5 mM) in a mM triethanolamine/HCl, mM MnCl2, mM dithiothreitol pH 7.7 buffer at 37 °C (4 mL, final volume) The carbohydrate phosphates were identified after dephosphorylation and acetylation by the usual method The acetylated crude residue was analysed by GCMS, and the analytical data compared with those of a synthetic reference of deoxyxylulose triacetate Reversibility of the formation of DXP from MEP by H-DXR MEP (10 mg) was treated overnight with H-DXR (1.2 mg) in the presence of NADP+ (0.5 mM) in a mM triethanolamine/HCl, mM MgCl2, mM dithiothreitol pH 7.7 buffer at 37 °C (4 mL final volume) NADP+ was regenerated using the acetone/alcohol dehydrogenase from Thermoanaerobium brockii [40] After dephosphorylation using an alkaline phosphatase, the mixture was lyophilized and acetylated Deoxyxylulose triacetate was isolated by TLC and identified by 1H-NMR [6] In other experiments, [5-13C]MEP or [2-13C]MEP (8 mM) was treated overnight with H-DXR (1.1 mg) in the presence of NADP+ (3 mM) in a 50 mM NH4HCO3, mM MgCl2 and mM dithiothreitol buffer at 37 °C (0.5 mL, final volume) NADP+ was regenerated using the acetone/ alcohol dehydrogenase from Thermoanaerobium brockii The reaction was directly performed in a NMR tube and monitored by 13C-NMR (50 MHz) using [2-13C]glycerol as internal reference (d ¼ 71.3 p.p.m.) Determination of the apparent equilibrium constant of the DXR reaction The assays were performed in a 50 mM Tris/HCl pH 7.5 buffer containing mM MnCl2 and mM dithiothreitol at 37 °C H-DXR (12 lg) was incubated in the presence of 0.116 mM MEP and NADP+ at different concentrations (0.088–0.352 mM) or at fixed concentration of NADP+ (0.176 mM) with MEP at different concentrations (0.058– 1.16 mM) The reactions were followed at 340 nm until the absorbance reached a plateau The concentration of produced NADPH was determined from the absorbance (e ¼ 6220 M)1 cm)1, kmax ¼ 340 nm) The influence of DXP (0.51–0.153 mM final concentration) or NADPH (6.9–20.4 lM final concentration) on the concentration of NADPH formed during the incubation of the enzyme with NADP+ (0.176 mM) and MEP (0.116 mM) was determined by the same UV absorption method 13 C-NMR study of the rearrangement reaction of H-DXR The reactions were directly performed in NMR tubes (5 mm diameter) in a 50 mM NH4HCO3 buffer containing mM MgCl2 and mM dithiothreitol at 37 °C The [1-13C]DXP concentration was 12.5 mM H-DXR (100 lg) was added to initiate the enzymatic reaction The influence of 0.5 mM NADP+, 0.5 mM ATP-ribose and 0.5 mM H2NADPH was tested The activity of the enzyme was demonstrated by adding NADPH (0.3 mM) and its regenerating system, isopropanol/alcohol dehydrogenase from Thermoanaerobium brockii The reaction medium (620 lL final volume) contained D2O (100 lL) and [2-13C]glycerol (1 mg) as an internal reference (d ¼ 71.3 p.p.m.) 13C- NMR spectra were recorded after h incubation The 13C chemical shifts of the possible metabolites resulting from the retro-aldol cleavage of DXP are hydroxyacetone 25 and glycoaldehyde phosphate 12 The 13C shifts of hydroxyacetone 25 (0.7 M) and glycoaldehyde phosphate 12 (0.3 M) were determined in the same medium Hydroxyacetone 25: 13 C-NMR (50 MHz, 50 mM NH4HCO3): d ¼ 24.0 (C-3, CH3), 66.7 (C-1, CH2OH), 211.0 (C-2, CO) Glycoaldehyde 2-phosphate 12: 13C-NMR (50 MHz, 50 mM NH4HCO3): d ¼ 66.0 (d, C-2, J ¼ 3.3 Hz, CH2OP), 88.4 [d, C-1, J ¼ 6.6 Hz, CH(OH)2] Kinetic studies of (3S )-3-hydroxypentan-2-one 5-phosphate 14 and (4S )-4-hydroxypentan-2-one 5-phosphate 19 with H-DXR H-DXR was incubated with (3R)-3-hydroxypentan-2-one 5-phosphate 14 (0.5 mM) or (4S)-4-hydroxypentan-2-one 5-phosphate 19 (0.5 mM) and NADPH (0.15 mM) in a 50 mM Tris/HCl, mM MnCl2, mM dithiothreitol pH 7.5 buffer The reaction was followed at 340 nm to observe the formation of NADP+ The inhibition of the enzymatic activity of DXR by (3S)-3-hydroxypentan-2-one 5-phosphate 14 and (4S)-4-hydroxypentan-2-one 5-phosphate 19 was studied by determining the influence of the two compounds [0.8–2.4 mM for (3S)-3-hydroxypentan-2-one 5-phosphate 14, 0.022–0.110 mM for (4S)-4-hydroxypentan-2-one 5-phosphate 19] on the enzymatic rate The concentration of DXP varied between 75 and 510 lM DXR (4 lg) was added last to initiate the reaction RESULTS AND DISCUSSION 2-C-Methyl-D-erythrose 4-phosphate as intermediate in the DXR-catalyzed reaction 2-C-Methyl-D-erythrose 4-phosphate was postulated as an intermediate in the first step of the reaction catalyzed by DXR It results from an a-ketol rearrangement of DXP and, after reduction, yields MEP From the analogy of the latter reaction sequence with that involved in the formation of the carbon skeleton of amino acids with a branched sidechains, aldehyde was expected to be only a transient intermediate not released from the enzyme active site, much like 3-hydroxy-3-methyl-2-oxobutyrate resulting from rearrangement of 2-acetolactate by acetohydroxy acid isomeroreductase (AHIR; EC 1.1.1.86) in the biosynthesis of branched-chain amino acids [42] For testing its possible role, unlabelled 2-C-methylD-erythrose 4-phosphate was synthesized by an adaptation of our former synthesis of MEP (Fig 2) [33] 2-O-Benzyl2-C-methyl-D-erythrose 4-dibenzylphosphate 13 was obtained as previously described in six steps from commercially available 1,2-O-isopropylidene-a-D-xylofuranose 26 Hydrogenolysis of the benzyl groups in methanol yielded the 2-C-methyl-D-erythrose 4-phosphate dimethylacetal, which upon hydrolysis with an acidic DowexÒ resin afforded 2-C-methyl-D-erythrose 4-phosphate The putative intermediate was tested as substrate of H-DXR assays and was utilized as reference material for the detection of this aldehyde in the DXR enzyme tests Preliminary kinetic studies were performed in order to determine the optimal conditions for the DXR-catalyzed Ó FEBS 2002 4452 J.-F Hoeffler et al (Eur J Biochem 269) enzymatic reaction Assays were performed in a triethanolamine/HCl buffer instead of the usual Tris/HCl buffer [9] because Tris is known to react with aldehydes [43] DXR requires divalent cations such as Mn2+, Co2+ or Mg2+ for its catalytic activity [9,16] For our enzyme system, maximal rates were found for mM Mn2+ and mM Mg2+ concentrations, indicating that the enzyme has more affinity for Mn2+ cations than for Mg2+ cations The usual concentration of NADPH is 0.3 mM [9] However, a 0.15 mM concentration was chosen for NADPH because higher concentrations resulted in lower rates When 2-C-methylD-erythrose 4-phosphate was incubated with H-DXR in the presence of NADPH, consumption of the cofactor was shown by the decrease in the absorption at 340 nm, suggesting that the enzyme reduced the aldehyde For the identification of MEP, the expected reaction product, the reaction mixture obtained after the reduction of 2-C-methylD-erythrose 4-phosphate with NADPH was dephosphorylated using an alkaline phosphatase and freeze-dried The crude residue was acetylated, and TLC allowed the isolation of methylerythritol triacetate, which was identified by NMR by comparison with a synthetic reference sample [44] This confirmed that 2-C-methyl-D-erythrose 4-phosphate was effectively reduced to MEP by H-DXR in the presence of NADPH Under these reaction conditions, deoxyxylulose triacetate could not be isolated after TLC The reverse reaction, isomerization of 2-C-methyl-D-erythrose 4-phosphate into DXP did not take place significantly in the presence of NADPH The formation of DXP from methylerythrose by H-DXR was, however, observed by incubating the aldehyde in the presence of NADP+ As methylerythrose and DX, as well as the diacetate of methylerythrose and the triacetate of DX, have the same Rf, the presence of DX triacetate was checked by GC and GCMS The retention times of the detected products were compared with those of synthetic 1-deoxy-D-xylulose triacetate In contrast with the almost quantitative formation of MEP from aldehyde 4, the formation of DXP was very low (% 7% yield as shown by GC detection of DX triacetate) Furthermore, GC-MS (chemical ionization with i-butane) of the acetylated crude reaction mixture showed a peak with the retention time of deoxyxylulose triacetate and characterized by a pseudo molecular ion at (M + H)+ (m/z ¼ 261) and by an ion corresponding to the loss of acetic acid from the deoxyxylulose triacetate (m/z ¼ 201) This confirmed the presence of small amounts of 1-deoxyD-xylulose triacetate The Km values measured for methylerythrose phosphate (294 lM in the presence of mM MnCl2 and 158 lM in the presence of mM MgCl2) for the E coli H-DXR were significantly higher than those found for DXP (73 lM for mM MnCl2 and 97 lM for MgCl2) and also depended on the nature of the divalent cation Despite several reproducible measurements, for unknown reasons the Km values we determined for DXP in the presence of MnCl2 (1 mM) differed significantly from those found in the literature for the same enzyme from E coli (Km ¼ 250 lM) [45] or from Z mobilis (Km ¼ 300 lM) [10] However, the Km values for DXP (Km ¼ 97 lM) when MgCl2 was used were similar to those published for the purified E coli enzyme wild-type (Km ¼ 99 lM) [45] and for S coelicolor DXP reductoisomerase (Km ¼ 60 lM) [13] The results obtained with an enzyme bearing a His-tag, like most those of the literature concerning His-tagged proteins, may not be directly extended to the native enzyme As the amino-terminal part of DXR is involved in the binding of the cofactor [46], the Histag, which is localized at the N-terminal end, may influence the enzymatic activity of H-DXR As for AHIR [42], the reduction step required the presence of a divalent metal cation, which may be involved in the binding of the aldehyde and/or the cofactor to the enzyme Whether such a metal cation is also required for the isomerization remains to be shown With DXP as a substrate, no significant difference of the kinetic constants was observed at optimal concentrations of Mn2+ (1 mM) and Mg2+ (3 mM) (Table 1) The influence of the nature of the divalent cation was, however, more pronounced for methylerythrose phosphate In the presence of Mn2+, the binding of the aldehyde was less efficient than in the presence of Mg2+ Indeed, although the chemical and biochemical behaviour of Mn2+ resembles that of Mg2+, ˚ ˚ Mn2+ (0.75 A) is somewhat larger than Mg2+ (0.65 A) In 2+ addition, Mn binds more readily to a site containing nitrogen in addition to oxygen than Mg2+, which prefers oxygen only [47] These peculiar properties of the two metal cations may influence the binding of methylerythrose phosphate to the active site, and thus explain the different Km values for the aldehyde The higher rate of reduction observed with Mn2+ could be due to a faster release of MEP, the reaction product Interestingly, the methyl group of methylerythrose phosphate is essential for the binding to the enzyme In our reaction conditions, D-erythrose 4-phosphate was neither a substrate, nor an inhibitor of the H-DXR (data not shown) It was recently reported that D-erythrose 4-phosphate is a poor substrate of DXR [17] According to our results, methylerythrose phosphate apparently has a good affinity with the enzyme, at least as compared with that of DXP Table Determination of the kinetic parameters (Km and V) of DXP and 2-C-methyl-D-erythrose 4-phosphate Assays were carried out in a 50 mM triethanolamine/HCl, mM MnCl2 (or mM MgCl2), mM dithiothreitol pH 7.7 buffer at a fixed concentration of NADPH (0.15 mM) The concentration of DXP varied from 31 to 310 lM while the concentration of 2-C-methyl-D-erythrose 4-phosphate varied from 93 to 620 lM The concentrations of the stock solutions of substrate were determined enzymatically using the H-DXR The enzyme (4.3 lg) was added last in order to initiate the reaction DXP 2-C-Methyl-D-erythrose 4-phosphate mM MnCl2 Km (lM) V (lmolỈmin)1Ỉmg DXR)1) mM MgCl2 mM MnCl2 mM MgCl2 73 10.5 97 10.5 294 20.6 158 11.9 Ó FEBS 2002 Deoxyxylulose phosphate reductoisomerase (Eur J Biochem 269) 4453 (Table 1) As erythrose 4-phosphate has a very weak affinity with the DXR, it appears that the methyl group at C-2 must play a crucial role for the binding of the substrate to the enzyme active site DXR and AHIR, an enzyme involved in the biosynthesis of branched-chain amino acids [42], catalyze similar reactions The latter converts 2-acetolactate or 2-aceto-2hydroxybutyrate into 2,3-dihydroxy-3-isovalerate or 2,3dihydroxy-3-methylvalerate This reaction proceeds in two steps: an isomerization, consisting of an alkyl migration, is followed by an NADPH-dependent reduction of the oxo group to give the final product In the reactions catalyzed by the two enzymes, the ketol-acid and the DXP isomeroreductase, the formation of the expected intermediates, 3-hydroxy-3-methyl-2-oxo-butyrate or methylerythrose phosphate, respectively, has never been shown For AHIR, it was suggested that the intermediate may be tightly bound to the enzyme or that the reduction takes place during the alkyl transfer so that the intermediate is never really formed [42] In none of our assays could the formation of methylerythrose phosphate be detected Assays designed to dissociate the transposition step from the reduction when using DXP as substrate were performed for a tentative direct identification of methylerythrose phosphate In the absence of cofactor, no isomerization was observed Accordingly, the simultaneous presence of the divalent cation and of the cofactor might be required for the subsequent fixation of DXP or methylerythrose phosphate [17] DihydroNADPH, an NADPH analogue [34] which is not a reducing cofactor, was expected to bind to the enzyme much like the natural coenzyme [17] Inhibition of the reaction by dihydro-NADPH would suggest that this analogue was bound to the active site of the enzyme It was, however, impossible to induce the isomerization of DXP into methyl erythrose phosphate The reduction step seems to represent the driving force to perform the rearrangement The fact that the postulated oxo intermediate in both isomeroreductase-catalyzed reactions are substrates for the reduction step with higher Km than those of the normal substrates suggests that the first proposition, their tight binding to the enzyme, is rather improbable The reduction step might be necessary in order that the isomerization takes place Reversibility of the DXR-catalyzed reaction: formation of DXP from MEP In order to verify that the H-DXR is capable of catalyzing the reverse reaction, the enzyme was incubated with MEP and NADP+ A first series of experiments was performed with 13C-labelled MEP When H-DXR was incubated in the presence of [5-13C]MEP or [2-13C]MEP and NADP+, a decrease of the C-5 (d ¼ 17.7 p.p.m.) or of the C-2 (d ¼ 73.2 p.p.m.) signals from MEP was observed, accompanied by a concomitant appearance and following increase of new signals corresponding to C-1 (d ¼ 25.5 p.p.m.) and C-2 (d ¼ 212.4 p.p.m.) from [1-13C]DXP or [2-13C]DXP, respectively A second experiment was performed with unlabelled MEP The increase of the absorbance at 340 nm, due to the formation of NADPH, suggested that MEP was at least oxidized to 2-C-methyl-D-erythrose 4-phosphate 4, the supposed intermediate of the reaction To show that the enzyme is capable of performing the two steps of the reverse process, converting MEP into DXP 3, i.e oxidation of MEP into and rearrangement of into DXP 3, MEP was treated with H-DXR in the presence of NADP+ and a regeneration system of the coenzyme to favour the reaction in the direction of the formation of DXP After dephosphorylation and acetylation of the reaction mixture, 1-deoxy-D-xylulose triacetate and 2-C-methylD-erythritol triacetate were isolated by TLC and identified by 1H-NMR, proving that H-DXR catalyzed the reverse transformation of MEP into DXP 3, including not only the oxidation of MEP to 2-C-methyl-D-erythrose 4-phosphate 4, but also the rearrangement of the latter aldehyde into DXP Methylerythrose diacetate, which coelutes with DX triacetate, was not observed When the reverse reaction with MEP (115 lM) and NADP+ (175 lM) was followed during several minutes the absorbance increase stopped completely when about 13–14% of the MEP was transformed, corresponding to the production of some NADPH (15.6 lM) The addition of more H-DXR to the reaction medium did not induce any additional absorbance increase Ceasing NADPH formation was not due to the inactivation of the enzyme, but rather to the fact that the equilibrium had been reached This low production of NADPH suggests that the reaction equilibrium is largely in favour of the production of MEP from DXP In order to confirm this hypothesis, the influence of the concentrations of NADP+ and MEP 5, the substrates of the reverse reaction, and of NADPH and DXP 3, the products of the reaction, on the total amount of NADPH produced were analysed The apparent equilibrium constant Kẳ ẵDXPeq ẵNADPHeq ẵHỵ ẵMEPeq ẵNADPỵ eq where [DXP], [MEP], [NADPH] and [NADP+] represent the concentrations of the different compounds at the equilibrium, was calculated in each case and found to be approximately the same (average value 4.6 ± 0.5 · 10)10 M at 37 °C) Attempts to determine the Km of MEP showed that the Kms of MEP (116 lM) and DXP (76 lM) had similar values The V of the reverse reaction (3.5 mMỈmin)1Ỉmg protein)1) was about 60% of that of the formation of MEP from DXP (5.6 mMỈmin)1Ỉmg protein)1) Even the very transitory existence of methylerythrose phosphate is no longer an assumption, as this aldehyde is the substrate of the DXP reductoisomerase with a good affinity It is converted into MEP 5, as well as into DXP Furthermore, our NMR data afforded direct evidence for the reversibility of the reaction catalyzed by the DXR by the conversion of MEP into DXP Rearrangement of DXP to 2-C-methyl-D-erythrose4-phosphate 4: the role of NADPH The conversion of DXP into MEP by the DXR requires the presence of NADPH as cofactor The first step of this conversion, i.e the rearrangement of DXP into methylerythrose phosphate 4, is, however, formally fully independent of this cofactor, as this rearrangement only corresponds to an isomerization In order to try to shed light on the possible role of methylerythrose phosphate 4, Ó FEBS 2002 4454 J.-F Hoeffler et al (Eur J Biochem 269) the DXR-catalyzed reaction was followed by 13C-NMR using a 13C-labelled substrate (99% isotope abundance) in order to improve the sensitivity of the detection method [1-13C]DXP was incubated in the presence of NADPH and of a NADPH regenerating system The enzyme preparation was active The decrease of the intensity of the C-1 signal of [1-13C]DXP (d ¼ 25.5 p.p.m.) was accompanied by the concomitant increase of the C-5 signal of [5-13C]MEP (d ¼ 17.7 p.p.m.) In a second experiment, NADPH was omitted in order to check whether the presence of the reducing cofactor is essential in the transposition step When [1-13C]DXP was incubated alone in the absence of NADPH, no additional 13C signal was observed, and especially no signal corresponding to the C-5 methyl group of methylerythrose phosphate (d ¼ 16.34 p.p.m., aldehyde form; d ¼ 18.25 p.p.m hydrate form), indicating that no reaction had occurred, at least within the limits of the 13 C-NMR detection The presence of the native coenzyme seems essential for the rearrangement, although it is not formally required In the presence of NADPH analogues, such as NADP+, dihydro-NADPH or ATP-ribose, DXP remained intact and no conversion into methylerythrose phosphate was observed In conclusion, direct evidence for the formation of 2-Cmethyl-D-erythrose 4-phosphate was not obtained in this enzymatic reaction, and no isomerization took place in the absence of NADPH These results were consistent with those of previous observations reported in the literature: the binding of NADPH first only allowed the binding of the DXP [17] Formation of 2-C-methyl-D-erythrose 4-phosphate from DXP: an a-ketol rearrangement or a retro-aldolization reaction? Incubation of [4,5-13C2]glucose into triterpenoids of the hopane series from Methylobacterium fujisawaense demonstrated for the first time the presence of a rearrangement in the MEP pathway [48] By analogy with the biosynthesis of the amino acids with branched side-chains, the conversion of DXP into MEP was considered as an a-ketol rearrangement (Fig 3) This mechanism involves the deprotonation of the hydroxyl group at C-3 of DXP 3, followed by the migration of the phosphate-bearing C2 subunit to afford methylerythrose phosphate Examples of reactions utilizing a retroaldolization/aldolization type mechanism in the place of an a-ketol rearrangement are also found in the literature, e.g the reaction catalyzed by the ribulose 5-phosphate 4-epimerase [49,50] In addition, such aldolization reactions often require the presence of a divalent cation [50], much like the reaction catalyzed by DXR For the formation of MEP from DXP 3, such an alternative mechanism would involve the deprotonation of the C-4 hydroxyl group of DXP 3, followed by the cleavage of the carbon–carbon bond between the carbon atoms C-3 and C-4 to give the enolate of hydroxyacetone 25 and glycoaldehyde phosphate 12 (Fig 3) Recombination of the two resulting moieties, rearranged via an aldolization by formation of a novel carbon–carbon bond between the carbon atoms derived from C-2 and C-4 of DXP 3, would give 2-C-methyl-D-erythrose 4-phosphate In order to try Fig Conversion of DXP into MEP by the DXR: a-ketol rearrangement vs retro-aldolization/aldolization Ó FEBS 2002 Deoxyxylulose phosphate reductoisomerase (Eur J Biochem 269) 4455 to get more insight into the mechanism of the DXRcatalyzed reaction, the DXP analogue 14 was synthesized and analysed for its behaviour towards the DXR On the one hand, if DXP analogue 14 is transformed to the (2R)-2-hydroxy-2-methylbutanol 4-phosphate 27, the reaction is most probably an a-ketol rearrangement On the other hand, the retro-aldolization would imply the intermediary formation of glycoaldehyde phosphate 12 and of the enol of hydroxyacetone 25 (Fig 3) Formation of 2-C-methyl-D-erythrose 4-phosphate from DXP: an a-ketol rearrangement? The possibility that DXR catalyzes the formation of 2-C-methyl-D-erythrose 4-phosphate from DXP via an a-ketol rearrangement (Fig 3) was checked by testing (3S)-3-hydroxypentan-2-one 5-phosphate 14 as potential substrate of H-DXR This compound was synthesized from the commercially available (S)-2-hydroxy-c-butyrolactone 15, which has the required configuration for the C-2 asymmetric carbon of 14 (Fig 4) Protection of the secondary alcohol of 15 afforded the silyl ether 16 in 97% yield Addition of methyl lithium gave a lactol, which opened under standard phosphorylation condition to the enantiomerically pure ketone 17, but with low yield [37] Deprotection of the silyl ether 17 with fluoride salts (TBAF), followed by hydrogenolysis of the benzyl groups, afforded (3R)-3-hydroxypentan-2-one 5-phosphate 14 Incubation of the DXP analogue 14 with H-DXR in the presence of NADPH did not induce any decrease of the absorbance at 340 nm The DXP analogue 14 was not a substrate of DXR It was, however, recognized by DXR and reversibly inhibited the enzyme as a mixed-type inhibitor (Ki ¼ 120 lM) In conclusion, no information was obtained on the reaction mechanism of the DXRcatalyzed reaction, but the crucial role of the C-4 hydroxy group of DXP was pointed out in the isomerization steps Furthermore, as the DXP analogue 14 inhibited the DXR, its isomer, (4S)-4-hydroxypentan-2-one 5-phosphate 19, was also synthesized and tested on H-DXR The synthesis started with the preparation of the silyl ether 21 from commercially available (R)-glycidol 20 (Fig 4) Epoxide opening of 21 with isoproprenylmagnesium bromide in the presence of CuI, followed by the deprotection of the silyl ether, gave diol 22 in excellent yield Selective phosphorylation of the primary alcohol of 22 with dibenzylphosphate chloride [37] at low temperature followed by oxidative cleavage [38] of the double bond yielded the protected ketone 23 in 39% yield over two steps Finally the benzyl groups were quantitatively removed by hydrogenolysis in the presence of a catalytic amount of palladium on activated carbon to yield (4S)-4-hydroxypentan-2-one 5-phosphate 19 (Fig 4) Analogue 19 also inhibited the DXR as a mixed type inhibitor (Ki ¼ 800 lM) Formation of 2-C-methyl-D-erythrose 4-phosphate from DXP: a retro-aldolization mechanism? In the case of an alternative retro-aldolization mechanism, hydroxyacetone 25 and glycoaldehyde phosphate 12 are the two intermediates leading to the formation of methylerythrose phosphate (Fig 3) Detection of hydroxyacetone 25 was attempted by incubation of [1-13C]DXP and following the reaction by 13 C-NMR spectroscopy No signal corresponding to the C-3 of hydroxyacetone 25 (d ¼ 24.0 p.p.m.), which was expected to be labelled in the case of hydroxyacetone Fig Synthesis of (3S)-3-hydroxypentan-2-one 5-phosphate 14 and (4S)-4-hydroxypentan-2-one 5-phosphate 19 (A) Synthesis of (3R)-3hydroxypentan-2-one 5-phosphate 14 (i) TBDMSOTf, 2,6-lutidine, dichloromethane, 97%; (ii) CH3Li, tetrahydofuran, )78 °C, 67%; (iii) (BnO)2POCl, pyridine, 16%; (iv) Bu4NF, tetrahydofuran, 88%; (v) H2, Pd/C, EtOH, 100% (B) Synthesis of (4S)-4-hydroxypentan-2one 5-phosphate 19 (i) TBDPSCl, imidazole, dichloromethane, 92%; (ii) CH2 ¼ CHMgBr, CuI, tetrahydofuran; (iii) Bu4NF, tetrahydofuran, 89% from two steps; (iv) (BnO)2POCl, pyridine, )40 °C, 54%; (v) RuCl3, NaIO4, CH3CN, CCl4, H2O, 72%; (vi) H2, Pd/C, EtOH, 100% formation, was observed next to those of C-1 of DXP (d ¼ 25.5 p.p.m.) and C-5 of MEP (d ¼ 17.7 p.p.m.) In addition, the influence of hydroxyacetone 25 and glycoaldehyde phosphate 12, the two intermediates in a retroaldolization/aldolization mechanism (Fig 3), on the activity of DXR was checked When the enzyme was incubated in the presence of the two compounds at concentrations up to mM, and NADPH, no decrease of the absorbance at 340 nm was observed, indicating that no MEP was produced In addition, the two compounds, either alone or together at concentrations of up to mM, did not inhibit the production of MEP from DXP They not seem to be recognized by DXR These negative results did not enable us to retain or exclude either one or the other mechanism The absence of NADPH consumption during the incubation of the enzyme Ó FEBS 2002 4456 J.-F Hoeffler et al (Eur J Biochem 269) with analogue 14 might be in favour of a retro-aldolization but this feature is better explained by the absence of formation of a productive complex between the analogue and the divalent ions Both DXP analogues, 14 and 19, each lacking a hydroxyl group either at C-4 or at C-3, respectively, inhibited the DXR These hydroxyl groups are apparently essential in the rearrangement reaction It is very likely that the hydroxyl groups are sites for the chelation of a divalent cation such as Mg2+ or Mn2+, most probably acting as Lewis acids facilitating the rearrangement reaction Furthermore the sequential mechanism followed by the enzyme has been confirmed: NADPH and the divalent cation bind first and precede the binding of the substrate [17] There is some evidence that this sequence implies a conformational change of the enzyme to allow the reaction as well for the isomerization step as for the reduction, as methylerythrose phosphate is a substrate of DXR More detailed analysis of the mechanism of 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5, the substrates of the reverse reaction, and of NADPH and DXP 3, the products of the reaction, on the total amount of NADPH... by the DXR by the conversion of MEP into DXP Rearrangement of DXP to 2-C-methyl-D-erythrose4 -phosphate 4: the role of NADPH The conversion of DXP into MEP by the DXR requires the presence of. .. demonstrated for the first time the presence of a rearrangement in the MEP pathway [48] By analogy with the biosynthesis of the amino acids with branched side-chains, the conversion of DXP into MEP