Báo cáo khoa học: Biosynthesis of isoprenoids – studies on the mechanism of 2C-methyl-D-erythritol-4-phosphate synthase pdf

Biosynthesis of isoprenoids – studies on the mechanism of 2C-methyl- D-erythritol-4-phosphate synthase Susan Lauw, Victoria Illarionova, Adelbert Bacher, Felix Rohdich and Wolfgang Eisenreich Center for Integrated Protein Research, Lehrstuhl fu ¨ r Biochemie, Department Chemie, Technische Universita ¨ tMu ¨ nchen, Garching, Germany Terpenes are the largest group of natural products, comprising more than 35 000 compounds [1]. They are all biosynthesized from two simple precursors, isopen- tenyl diphosphate and dimethylallyl diphosphate. These universal precursors were initially believed to be biosynthesized exclusively via the mevalonate pathway [2–4], but more recent studies have shown the existence of a second pathway via 1-deoxy-d-xylulose 5-phos- phate (1) and 2C-methyl-d-erythritol 4-phosphate ( 3) (Fig. 1) [5–9]. This pathway is now known to supply the precursors for the isoprenoids of apicomplexan protozoa and of many eubacteria, as well as for the majority of isoprenoids from plants [10–14]. 2C-Methyl-d-erythritol-4-phosphate synthase (IspC), encoded by the ispC gene (also designated dxr), cata- lyzes the first committed step in the nonmevalonate pathway [15] and has been shown to be the molecular target of fosmidomycin [16,17], an antibiotic from Keywords deoxyxylulose; dimethylallyl diphosphate; isopentenyl diphosphate; terpene Correspondence W. Eisenreich, Center for Integrated Protein Research, Lehrstuhl fu ¨ r Biochemie, Department Chemie, Technische Universita ¨ t Mu ¨ nchen, Lichtenbergstr. 4, D-85747 Garching, Germany Fax: +49 89 289 13363 Tel: +49 89 289 13336 E-mail: wolfgang.eisenreich@ch.tum.de F. Rohdich, Center for Integrated Protein Research, Lehrstuhl fu ¨ r Biochemie, Department Chemie, Technische Universita ¨ t Mu ¨ nchen, Lichtenbergstr. 4, D-85747 Garching, Germany Fax: +49 89 289 13363 Tel: +49 89 289 13336 E-mail: felix.rohdich@ch.tum.de (Received 11 March 2008, revised 8 June 2008, accepted 11 June 2008) doi:10.1111/j.1742-4658.2008.06547.x 2C-Methyl-d-erythritol-4-phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalo- nate isoprenoid biosynthetic pathway. The reaction involves the isomeriza- tion of 1-deoxy-d-xylulose 5-phosphate, giving a branched-chain aldose derivative that is subsequently reduced to 2C-methyl-d-erythritol 4-phos- phate. The isomerization step has been proposed to proceed as an intramo- lecular rearrangement or a retroaldol–aldol sequence. We report the preparation of 13 C-labeled substrate isotopologs that were designed to opti- mize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol–aldol reaction sequence. In reaction mixtures containing large amounts of 2C-methyl-d-erythritol-4-phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidop- sis thaliana, and a mixture of [1- 13 C 1 ]-2C-methyl-d-erythritol 4-phosphate and [3- 13 C 1 ]2C-methyl-d-erythritol 4-phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorpo- rated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1-deoxy-d-xylulose 5-phosphate to give 1-deoxy-l-ribulose 5-phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol–aldol mechanism cannot be ruled out conclusively, the data show that a retrol- dol–aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three geneti- cally distant organisms. Abbreviation IspC, 2C-methyl- D-erythritol-4-phosphate synthase. 4060 FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS Streptomyces lavendulae [18,19]. The development of that compound as an antibiotic drug was aborted in the 1980s, but recent work has shown activity against various Plasmodium spp., including Plasmodium fal- ciparum, a major human pathogen [17,20–22]. These studies have validated IspC as a target for the develop- ment of novel antimalarial agents, which are urgently needed in light of the enormous death toll of malaria [23] and the rapid dissemination of variants with resis- tance against currently available drugs [24]. Moreover, IspC and the consecutive enzymes of the pathway are believed to be potential targets for the chemotherapy of infections by a variety of eubacterial pathogens, most notably Mycobacterium tuberculosis [14,25–27]. The first step of the reaction catalyzed by IspC has been shown to give the branched aldose derivative, 2C-methyl-d-erythrose 4-phosphate, which is subse- quently reduced to 2C-methyl-d-erythritol 4-phosphate [28]. The reductive reaction step has been shown to involve the transfer of a hydride ion from the pro-S position at C-4 of NADPH to the RE position of C-1 of reaction intermediate 2 (Fig. 1) [29,30]. The forma- tion of 2 from the linear deoxyketose-type substrate has been shown to proceed by cleavage of the bond between C-3 and C-4 and the generation of a novel bond between C-1 and C-3 of the substrate [31,32]. A sigmatropic rearrangement and a retroaldol–aldol reac- tion sequence are both compatible with the presently available data (Fig. 2) [28,31–34], whereas a hydride shift mechanism has been ruled out by isotope labeling studies [35]. Recently, the formation of 1-deoxy-l-ribu- lose 5-phosphate, an epimer of 1, was reported in an IspC-catalyzed reaction without NADPH and Mg 2+ or Mn 2+ , and this observation was interpreted as evidence for a retroaldol mechanism of the reaction catalyzed by IspC [36]. Here, we report on extensive stable isotope experiments aimed at discrimination between a sigmatropic rearrangement and a retro- aldol–aldol mechanism. Additional mechanistic infor- mation on the enzyme-catalyzed reaction could benefit the development of novel inhibitors for use as anti- infective drugs. Results IspCs of Escherichia coli, M. tuberculosis and Arabid- opsis thaliana were selected for parallel enzyme studies, after a phylogenetic analysis of 31 IspC amino acid Fig. 1. Reactions catalyzed by IspC: 1, 1-deoxy- D-xylulose 5-phosphate; 2, 2C-methyl- D-erythrose 4-phosphate; 3, 2C-methyl- D-erythritol 4-phosphate. Fig. 2. Hypothetical mechanism of the enzymatic reaction catalyzed by IspC: 1,1- deoxy- D-xylulose 5-phosphate; 2,2C-methyl- D-erythrose 4-phosphate; 4, glycolaldehyde phosphate; 5, enolate of hydroxyacetone. S. Lauw et al. Mechanism of IspC protein FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS 4061 sequences from prokaryotic and eukaryotic species had shown the genetic distance between these three enzyme species to be relatively large (Fig. 3). The degrees of sequence identity of the enzyme from E. coli with the orthologous enzymes from M. tuberculosis and A. tha- liana are 40% and 43%, respectively. Notably, the study organisms are located in three different branches of the dendrogram. As opposed to a sigmatropic rearrangement, a retro- aldol–aldol reaction sequence can involve the exchange of fragments between different substrate molecules, unless the reaction proceeds in strict containment in a reaction cavity that does not permit the escape and reutilization of reaction intermediates. The hypotheti- cal retroaldol cleavage of 2C-methyl-d-erythritol 4-phosphate (3), according to Fig. 4, should give glycolaldehyde phosphate (4) and hydroxyacetone (5) as reaction intermediates [33]. In order to measure the frequency of any potential intermediate exchange, we prepared two substrate isotopomers of 3 that were specifically designed to maximize the sensitivity for the diagnosis of fragment exchange by 13 C-NMR spectroscopy. Specifically, [1- 13 C 1 ]2C-methyl-d-erythri- tol 4-phosphate and [3- 13 C 1 ]2C-methyl-d-erythritol 4-phosphate (3a and 3b, respectively, Fig. 4) were obtained from [3,4- 13 C 2 ]glucose and [2,5- 13 C 2 ]glucose, respectively, by the enzyme-assisted one-pot reaction strategy described previously [37]. An enzyme-mediated recombination of fragments 4, 5a, 4a and 5 generated from a mixture of [1- 13 C 1 ]2C-methyl-d-erythrose 4-phosphate and [3- 13 C 1 ]2C-methyl-d-erythrose 4-phos- phate (2a and 2b, respectively) via the proposed retroaldol–aldol mechanism should result in the forma- tion of four isotopolog species of 1-deoxy-d-xylulose 5-phosphate (1a–1d, Fig. 4). Notably, the enzyme- mediated recombination of [1- 13 C 1 ]glycolaldehyde (4a) and the enolate of [1- 13 C 1 ]hydroxyacetone (5a) could then give [3,4- 13 C 2 ]1-deoxy-d-xylulose 5-phosphate Fig. 3. Phylogenetic tree of IspCs from vari- ous organisms. The consensus cladogram was constructed by neighbor-joining analysis from an alignment of IspC amino acid sequences from six plant species, one cya- nobacterium (Synechocystis sp.), one protist (P. falciparum), and 23 eubacteria represent- ing different families. Gaps were removed from the alignment, and the total number of positions taken into account was 327. The numbers at the nodes are the statistical confidence estimates computed by the bootstrap procedure. The bar represents 0.134 percent accepted mutation distance. Mechanism of IspC protein S. Lauw et al. 4062 FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS B C D A p.p.m. Fig. 4. NMR analysis of IspC assays using [1- 13 C 1 ]2C-methyl-D-erythritol 4-phosphate and [3- 13 C 1 ]2C-methyl-D-erythritol 4-phosphate (3a and 3b, respectively) as initial substrates. 1a, [3- 13 C 1 ]1-deoxy-D-xylulose 5-phosphate; 1b, [4- 13 C 1 ]1-deoxy-D-xylulose 5-phosphate; 1c, [3,4- 13 C 2 ]1- deoxy- D-xylulose 5-phosphate; 2a, [1- 13 C 1 ]2C-methyl-D-erythrose 4-phosphate; 2b, [3- 13 C 1 ]2C-methyl-D-erythrose 4-phosphate, 4, protonated glycolaldehyde phosphate (unlabeled); 4a, protonated [1- 13 C 1 ]glycolaldehyde phosphate; 5, enolate of hydroxyacetone (unlabeled); 5a, enolate of [1- 13 C 1 ]hydroxyacetone. 13 C-NMR signals of an incubation mixture without enzyme (A), and with IspC from E. coli (B), M. tuberculosis (C) and A. thaliana (D), respectively. The asterisks denote signals due to impurities. S. Lauw et al. Mechanism of IspC protein FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS 4063 (1c). This double-labeled species would be detected via satellite lines in the 13 C-NMR spectrum due to 13 C 13 C coupling. In order to maximize the diagnostic sensitivity, we decided to conduct the experiments under steady-state conditions where reactants 1 and 3 are present in simi- lar amounts at thermodynamic equilibrium. For that purpose, reaction mixtures containing 5 mm 3a,5mm 3b, 215 mm NADP + and 0.15–0.25 mm IspC from E. coli, M. tuberculosis or A. thaliana were incubated at pH 8 and 37 °C for 24 h and were monitored by 13 C-NMR spectroscopy. The partial reduction of NADP + by the enzyme rapidly resulted in steady-state conditions where the steady-state concentrations of 1 and 3 were approximately equal (Fig. 4). Conse- quently, the forward and the reverse reaction rate under equilibrium condition were also bound to be approximately equal. Notably, the IspC enzymes were present in very high (near-stoichiometric) concentra- tions. Under these conditions, the substrate molecules should be engaged by enzyme molecules on a near- permanent basis. The residual enzyme activity after 24 h of incubation was measured after massive dilution of an aliquot of the reaction mixture, using 1 as substrate. The decrease in activity during the 24 h incubation period was in the range 27–37% for the three different enzymes under study. From the starting conditions and the enzyme stabil- ity measurements under our reaction conditions, it follows that an average substrate molecule should have passed through approximately 8800, 12 100 and 2400 forward–reverse cycles in the experiments with enzymes from E. coli, M. tuberculosis and A. thaliana, respectively (supplementary Table S4). For the equilibrium constant of the reaction cata- lyzed by IspC as defined by Eqn (1), we obtained a value of (2.8 ± 0.2) · 10 )10 m at pH 8.0 and 37 °C. This is well in line with a value of (4.6 ± 0.5) · 10 )10 m at pH 7.7 and 37 °C that had been reported earlier [28]. K ¼ ½NADPH eq Á½1 eq Á½H þ  ½NADP þ  eq Á½3 eq ð1Þ Figure 4 shows 13 C-NMR signals of the reaction mixtures prior to the addition of enzyme (Fig. 4A) and after incubation with enzymes from E. coli (Fig. 4B), M. tuberculosis (Fig. 4C) and A. thaliana (Fig. 4D), respectively. Reaction mixtures treated with enzymes from the three different organisms studied showed very similar results. The crucial observation is the absence of any detect- able excess of the 13 C 13 C coupling satellites beyond the natural abundance level for the signals of C-3 and C-4 of a hypothetical product 1c. The hypothetical posi- tions of the 13 C 13 C coupling satellites expected in the spectrum of 1c are marked by arrows in Fig. 4B–D. In each case, the integrals of the satellite signals are in the range of 1% as compared to the central signal. Signals of that size would be expected in the complete absence of fragment exchange, where they reflect the presence of about 1.1% 13 C in those carbon atoms of the reactant that were not labeled. On the basis of the quantitative evaluation of the 13 C-NMR signal intensities and coupling satellites in experiments with 13 C-labeled substrates, it can be esti- mated that fewer than one fragment exchange has occurred during more than 100 000 reaction cycles. Although these data are not sufficient to rule out a ret- roaldol–aldol reaction sequence, they do show that a hypothetical retroaldol–aldol sequence would require extremely tight confinement of the intermediary molec- ular fragments at the active site of the enzyme. The limit for escape and reutilization of a retroaldol frag- ment would be fewer than once in 100 000 forward– reverse cycles. In this context, it is also worth noting that the branched intermediate 2C-methyl-d-erythrose 4-phosphate (2) (Fig. 1) can be used as substrate by the enzyme at a rate that is comparable with the con- version rate of substrate 1 [28]; thus, strict confinement seems at least not to apply to that intermediate. In a second set of experiments, we checked whether exogenous hydroxyacetone, whose enolate is the pre- dicted intermediate of the hypothetical retroaldol–aldol mechanism, can be incorporated into reactants 1 and 3 by fragment exchange. Preliminary experiments had shown that hydroxyacetone does not significantly change the catalytic rate of 2C-methyl-d-erythritol 4-phosphate synthase when present in concentrations up to 2% (v ⁄ v). The reaction mixtures contained 10 mm [1,3,4- 13 C 3 ]2C-methyl-d-erythritol 4-phosphate (3c, Fig. 5), 215 mm NADP + , 243 mm (2%, v ⁄ v) hydroxyacetone, 100 mm Tris ⁄ HCl (pH 8.0), and 0.23–0.25 mm IspC from E. coli, M. tuberculosis or A. thaliana. They were incubated for 8 h at 37 °C and were then analyzed by NMR spectroscopy. As described above, these initial conditions were rapidly conducive to steady conditions where 1 and 3 were present in very similar concentrations, and the rates of the forward reaction (conversion of 1 to 3) and the backward reaction (conversion of 3 into 1) were also essentially the same. Any ‘wash-in’ of unlabeled hydroxyacetone (6) should give the iso- topolog 1f, which carries only two 13 C atoms. This Mechanism of IspC protein S. Lauw et al. 4064 FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS A B C D p.p.m. Fig. 5. NMR analysis of IspC assays using [1,3,4- 13 C 3 ]2C-methyl-D-erythritol 4-phosphate and unlabeled hydroxyacetone (3c and 6) as initial substrates. 1e, [3,4,5- 13 C 3 ]1-deoxy-D-xylulose 5-phosphate; 1f, [4,5- 13 C 2 ]1-deoxy-D-xylulose 5-phosphate; 3d, [3,4- 13 C 2 ]2C-methyl-D-erythritol 4-phosphate; 4b, protonated [1,2- 13 C 2 ]glycolaldehyde phosphate; 5, enolate of hydroxyacetone (unlabeled); 5a, enolate of [1- 13 C 1 ]hydroxyace- tone; 6, hydroxyacetone (unlabeled). 13 C-NMR signals of an incubation mixture without enzyme (A), and with IspC from E. coli (B), M. tuber- culosis (C) and A. thaliana (D), respectively. The asterisks denote signals due to impurities. S. Lauw et al. Mechanism of IspC protein FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS 4065 isotopolog would be diagnosed easily in the 13 C-NMR spectra by a distinctive double doublet signature of C-4 that would be caused by 13 C 13 C coupling and 13 C 31 P coupling (predicted signal positions are indi- cated by arrows in Fig. 5B–D). Figure 5A–D shows 13 C-NMR signals detected in the exchange experiment with unlabeled hydroxyacetone. Signal intensities showed a steady-state ratio of 59 : 41 for 1 and 3c (supplementary Table S6). The crucial double doublets as expected for a retroaldol–aldol mechanism (Fig. 5B–D) were absent. The 13 C NMR data of 1e and 3c are shown in supplementary Table S5. In the following set of experiments, we investigated whether glycolaldehyde phosphate and hydroxyacetone can serve as direct substrates for IspC from different organisms to form 2C-methyl-d-erythritol 4-phosphate, as shown in the hypothetical retroaldol–aldol reaction sequence illustrated in Fig. 5. Specifically, the reaction mixtures contained 2 mm [1,2- 13 C 2 ]glycolaldehyde phosphate (4b), 243 mm (2%, v ⁄ v) hydroxyacetone (5), Tris ⁄ HCl (pH 8.0), 3 mm NADPH, and 0.21–0.34 mm IspC from E. coli, M. tuberculosis or A. thaliana. The 13 C-NMR spectra obtained after incubation periods of 1.5 and 3 h, respectively, showed only dou- ble doublet signals at 89.2 p.p.m. due to the presence of the hydrate of 4b (Fig. 6B–D). As shown in Fig. 6, no evidence for the formation of [3,4- 13 C 2 ]2C-methyl- d-erythritol 4-phosphate (3d) could be obtained. Nota- bly, it would have been possible to detect any 3d by the specific double doublet signature of C-3, as confirmed by a titration experiment with [1,3,4- 13 C 3 ]2C-methyl-d- erythritol 4-phosphate (3c) (Fig. 6E). It should be noted that these experiments were conducted with very high concentrations of enzymes (almost in the millimo- lar range) and with a very high concentration of 243 mm (2%, v ⁄ v) of hydroxyacetone, which had been shown to be tolerated by the enzyme without significant reduction in the rate for the IspC reaction measured with 1e as substrate (see also supplementary Table S7). Wong & Cox [36] reported the formation of 1-deoxy-l-ribulose 5-phosphate (7b, Fig. 7), an epimer of 1, in an IspC reaction mixture in the absence of NADPH and of divalent metal ions. Specifically, they observed a new 13 C-NMR signal at 71.6 p.p.m., which A B C D E p . p .m. Fig. 6. NMR analysis of IspC assays using protonated [1,2- 13 C 2 ]glycolaldehyde phos- phate and the enolate of hydroxacetone (4b and 5a, respectively) as initial substrates. 2c, [3,4- 13 C 2 ]2C-methyl-D-erythrose 4-phos- phate; 3d, [3,4- 13 C 2 ]2C-methyl-D-erythritol 4-phosphate. (A)–(E) are 13 C-NMR spectra obtained from IspC reactions using [1,2- 13 C 2 ]glycolaldehyde phosphate (4b) and hydroxacetone (5a) as substrates. 13 C-NMR signals of an incubation mixture without enzyme (A), and with IspC from E. coli (B), M. tuberculosis (C) and A. thaliana (D) and with the addition of [1,3,4- 13 C 3 ]2C-methyl-D- erythritol 4-phosphate after 3 h of incubation of the reaction mixture B (E). The asterisks denote signals due to impurities. Mechanism of IspC protein S. Lauw et al. 4066 FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS was assigned to C-4 of 7. When we repeated that experiment with [3,4,5- 13 C 3 ]-1 as substrate, we found that a signal was already present at 71.6 p.p.m., even prior to incubation of the reaction mixture (Fig. 8, lane A), and the intensity of that signal increased by a factor of about 2 during the subsequent incubation. Notably, the same phenomenon was observed in sam- ples without IspC. This unexpected finding prompted a more detailed investigation, which revealed that 1 is subject to spontaneous isomerization to give 7. The details are described below. Specifically, we prepared [3,4,5- 13 C 3 ]-1 by an enzy- matic procedure starting from [U- 13 C 6 ]glucose [37]. Despite the absence of IspC, the formation of the target compound 1 was accompanied by the formation of a compound characterized by a 13 C resonance at 71.6 p.p.m., albeit at a much lower rate. Specifically, after incubation for 1 h, the yield of 1 was about 50%, and the relative yield, based on 1, of the compound resonating at 71.6 p.p.m. was 1%. Even after the removal of all proteins by ultrafiltration, the relative amount of that contaminant continued to increase over a period of about 1 week; the final ratio of the two compounds, believed to represent a state of equilib- rium, was about 4 : 1. The apparent rate constant for the formation of 7 from 1 was 3 · 10 )7 s )1 , and, the equilibrium constant was calculated to be 3.45 (supple- mentary Fig. S1). In parallel experiments with and without addition of IspC, the 13 C signal at 71.6 p.p.m. increased at the same rate. More specifically, reaction mixtures containing 100 mm Tris ⁄ HCl (pH 8), 10 mm 1, and 0.1 mm (5 mgÆmL )1 ) IspC when required, were incubated at 37 °C. The component resonating at 71.6 p.p.m. increased from 3% to 5% (based on the concentration of 1) during a period of 24 h at 37 °C, irrespective of the presence or absence of IspC. Using an equilibrium mixture of [U- 13 C 5 ]-1 and of the component resonating at 71.6 p.p.m., we could assign all 13 C signals of the latter on the basis of 13 C 13 C and 13 C 31 P coupling in one-dimensional 13 C- NMR spectra (supplementary Fig. S2). All 1 H-NMR signals of the newly formed compound were then assigned by HMQC spectroscopy (supplementary Table S8). The NMR data were in close correspon- dence with those reported earlier for a chemically synthesized sample of 1-deoxy-l-ribulose 5-phosphate (7b, Fig. 7) [38]. However, it should be noted that 13 C-NMR is unable to discriminate between the d-enantiomer and l-enantiomer under the experimental conditions used, and enantiomer assignment of the 1-deoxyribulose 5-phosphate formed by spontaneous isomerization of 1-deoxy-d-xylulose 5-phosphate (1)is not possible from our experimental data. Discussion The main part of the present study was a search, under conditions of maximal stringency, for fragment exchange that could be the hallmark of the hypotheti- cal retroaldol–aldol mechanism. The essentials of that high-stringency strategy can be summarized as fol- lows: (a) the experiments shown in Figs 4 and 5 were conducted under steady-state conditions (at thermody- namic equilibrium), thus enabling each molecule to pass through thousands of forward–backward reaction cycles; (b) enzymes were used at near-stoichiometric concentrations, in order to engage substrate molecules on a near-permanent basis; (c) multiple 13 C labeling was used in order to optimize detection of the crucial molecular species that would have resulted from frag- ment exchange by the utilization of 13 C 13 C coupling; (d) substrates used included not only the natural sub- strate of the reaction, 1-deoxy-d-xylulose 5-phosphate (1), but also the hypothetical fragments that would be expected to result from a retroaldol fragmentation, i.e. hydroxyacetone and glycolaldehyde phosphate – one of these substrates (glycolaldehyde phosphate, 4b) was double-labeled with 13 C, and the other was used at an unusually high concentration (in the decimolar Fig. 7. Stereoisomers of 1-deoxy-D-xylulose 5-phosphate: 1, 1-deoxy- D-xylulose 5-phosphate; 7a, 1-deoxy-D-ribulose 5-phos- phate; 7b, 1-deoxy- L-ribulose 5-phosphate; 8, 1-deoxy-L-xylulose 5-phosphate. S. Lauw et al. Mechanism of IspC protein FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS 4067 range) in order to maximize diagnostic sensitivity; (e) three IspC orthologs from genetically distant sources were used, with the expectation that all orthologs would not necessarily confine fragments with the same degree of stringency – as it is unlikely that strong selective pressure specifically enforced very high degrees of stringency, it would not appear implausible that different taxa might have enzymes with different stringencies. On the basis of these results, the limit on observed fragment escape and fragment reutilization is fewer than one in many thousands of forward–reverse cycles. The active site of IspC is located close to the sur- face. A flexible loop at the active site (amino acids 206–216) [39–42] is able to fold into at least three dif- ferent conformations. Specifically, in the apoenzyme structure, this loop is unordered, whereas the structure with bound NADPH, and especially the complex with bound NADPH as well as 1-deoxy-d-xylulose 5-phos- phate (1), showed this loop to be well ordered and to be closing the active site region of the enzyme (Fig. 9). On the other hand, it has also been demonstrated that the branched intermediate 2 can access the active site cavity from the bulk solvent and can then serve as a substrate, obviously without hindrance from the said loop [28]. The available structural data are not a suffi- cient basis to support a claim of absolute confinement of the active site. Notably, both hypothetical fragments resulting from retroaldol cleavage would be small by comparison with the branched aldose intermediate 2; as the active site is even accessible to 2, one would expect that the hypothetical intermediates 4b and 5, which are both small by comparison, should be able to exchange with the bulk solvent. 3-Deoxy-1 and 4-deoxy-1 have been shown to act as weak inhibitors, but not as substrates of IspC [28,38]. Had these investigations resulted in the demonstration of any (even low) substrate activity for the 4-deoxy compound, that would have ruled out the retroaldol mechanism. Clearly, however, the reverse argument would be a logical fallacy; the failure of the 4-deoxy compound to act as a substrate could be due to a wide variety of reasons, and does not determine the mechanism. The claimed conversion of 1 into the epimer 7 by IspC in the absence of pyridine nucleotides and diva- lent metal ions could have been construed as support for a retroaldol mechanism. Unfortunately, our results suggest that the formation of 7 in those experiments was incorrectly ascribed to the catalytic action of IspC, and reflected, in reality, the spontaneous, uncatalyzed epimerization of 1. In summary, our data are all consistent with a sig- matropic rearrangement, albeit they do not constitute definite proof. However, it appears safe to say that the present experiments extend the degree of stringency to the limits of experimental feasibility as ultimately defined by the long-term chemical stability of the proteins, substrates and coenzymes involved. p . p .m. Fig. 8. 13 C-NMR spectra of 1-deoxy-D-xylu- lose 5-phosphate and its diastereomer. Spectra were recorded in time intervals of 2 days at 37 °C. (A) Day 0. (B) Day 2. (C) Day 4. (D) Day 6. (E) Day 8. The signals of [3,4,5- 13 C 3 ]1-deoxy-D-xylulose 5-phosphate (1e) are shown in orange, and those of [3,4,5- 13 C 3 ] 1-deoxy-L-ribulose 5-phosphate (7b) in green. Mechanism of IspC protein S. Lauw et al. 4068 FEBS Journal 275 (2008) 4060–4073 ª 2008 The Authors Journal compilation ª 2008 FEBS Experimental procedures Materials [U- 13 C 6 ]Glucose was purchased from Isotec, Miami Town- ship, OH; [3,4- 13 C 2 ]-glucose and [2,5- 13 C 2 ]glucose were from Omicron Inc., South Bend, OH. [3,4,5- 13 C 3 ]1-deoxy- d-xylulose 5-phosphate, [U- 13 C 5 ]1-deoxy-d-xylulose 5-phos- phate and [1,3,4,- 13 C 3 ]2C-methyl-d-erythrose 4-phosphate were synthesized as described previously [37,43,44]. Hexokinase from yeast, triosephosphate isomerase from rabbit muscle, glutamate dehydrogenase from bovine liver and glucose dehydrogenase from Thermoplasma acido- philum were from Sigma. 1-Deoxy-d-xylulose-5-phosphate synthase from Bacillus subtilis and 2C-methyl-d-erythritol 4-phosphate synthase (IspC) from E. coli were prepared by published procedures [37,43,44]. The preparation of IspC from A. thaliana, fructose-1,6-biphosphate aldolase, phosphofructokinase, glucose-6-phosphate isomerase from E. coli, 6-phosphogluconate dehydrogenase and glucose-6- phosphate dehydrogenase from B. subtilis is described elsewhere [44–47]. The recombinant proteins used for substrate synthesis and enzyme assays are listed in supple- mentary Table S3. Construction of a recombinant strain for hyperexpression of the M. tuberculosis ispC gene The ispC gene of M. tuberculosis (accession no. gb BX842581.1) was amplified by PCR using the oligonucle- otides ispCMycSacivo and ispCMycPstIhi as primers and chromosomal M. tuberculosis DNA as template. The amplifi- cate was digested with the restriction endonucleases SacI and PstI, and the resulting fragment was ligated into the expres- sion vector pQE30, which had been digested with the same restriction enzymes. The ligation mixture was electroporated into E. coli XL1-Blue [48] cells, giving the recombinant strains XL1-pQEispCMyco and M15-pQEispCMyco. Bacterial strains, plasmids and oligonucleotides used in this study are listed in supplementary Tables S1 and S2. Sequence determination DNA sequencing was performed by the automated dide- oxynucleotide method. N-terminal peptide sequences were obtained by pulsed-liquid mode. Expression of recombinant IspC from M. tuberculosis The recombinant E. coli strain XL1-pQEispCMyco was grown in LB broth containing ampicillin (180 mgÆL )1 )as appropriate. Cultures were incubated at 37 °C with shaking. At an attenuance of 0.7 (600 nm), isopropylthiogalactoside was added to a final concentration of 0.5 mm, and the cultures were incubated overnight at 30 °C. The cells were harvested by centrifugation at 5000 g for 30 min at 4°Con an SLA-3000 rotor (Sorvall, Du Pont, Newton, CT), washed with 0.9% (w ⁄ v) sodium chloride, and stored at )20 °C. Preparation of recombinant IspC from M. tuberculosis The frozen cell mass (40 g) of the recombinant E. coli strain XL1-pQEispCMyco was thawed in 200 mL of 100 mm Tris ⁄ HCl (pH 8.0), containing 0.5 m sodium chloride, 20 mm imidazole hydrochloride, and 10% (v ⁄ v) glycerol. The cells were disrupted using a French press, and the suspension was centrifuged at 15 000 g for 30 min at 40°C on an SS-34 rotor (Sorvall). The supernatant was applied to a column of Ni-chelating Sepharose FF (column volume, 34 mL) that had been equilibrated with 100 mm Tris ⁄ HCl (pH 8.0) containing 0.5 m sodium chloride, 20 mm imidazole hydrochloride, and 10% (v ⁄ v) glycerol (flow rate, 3 mLÆmin )1 ). The column was washed with 100 mm Tris ⁄ HCl (pH 8.0) containing 0.5 m sodium A B Fig. 9. Crystal structures of monomeric IspC from E. coli. (A) Apoenzyme (Protein Data Bank file 1K5H [41]). (B) Enzyme in com- plex with NADPH (orange) and 1-deoxy- D-xylulose 5-phosphate (blue) (Protein Data Bank file 1Q0Q [39]). The flexible loop (residues 206–216) in both structures is shown in magenta. S. Lauw et al. 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ETH Zurich, Zurich ¨ ¨ Mechanism of IspC protein 8 Eisenreich W, Menhard B, Hylands PJ, Zenk MH & Bacher A (1996) Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin Proc Natl Acad Sci USA 93, 643 1–6 436 9 Lichtenthaler HK, Schwender J, Disch A & Rohmer M (1997) Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway... addition of 1 m NaOH, and 300 U of hexokinase, 25 U of 4070 6-phosphogluconate dehydrogenase, 62 U of glucose-6-phosphate dehydrogenase and 100 U of glutamate dehydrogenase were added The mixture was incubated at 37 °C, and the reaction was monitored by 13C-NMR spectroscopy After 1 h, protein was removed by ultrafiltration (3 kDa cutoff) The solution was lyophilized The residue was dissolved in 6 mL of. .. and the pH was adjusted to 6 Sodium metaperiodate was added to a final concentration of 333 lm, and the mixture was incubated at room temperature and monitored by 13C-NMR After 10 min, the excess of periodate was quenched by the addition of glycerol to a final concentration of 1 mm, and [1,2-13C2]glycolaldehyde phosphate was purified on Dowex (Cl)-form, 3 g; volume, 10 mL) Spectrophotometric assay of the. .. substrate synthesis and enzyme assays Table S4 Conversion ratios and equilibrium constants for the IspC reaction Table S5 13C-NMR data of [3,4,5-13C3]1-deoxy-dxylulose 5-phosphate (1e) and [1,3,4-13C3]2C-methyl-derythritol 4-phosphate (3c) Table S6 Conversion ratios and cycles for the IspC reaction containing hydroxyacetone Table S7 Calculated hypothetical cycles for the IspC reaction containing [1,2-13C2]glycolaldehyde... 2 Qureshi N & Porter JW (1981) Conversion of acetylcoenzyme A to isopentenyl pyrophosphate In Biosynthesis of Isoprenoid Compounds (Porter JW & Spurgeon SL, eds), pp 4 7–9 4 John Wiley, New York, NY 3 Bloch K (1992) Sterol molecule: structure, biosynthesis, and function Steroid 57, 37 8–3 83 4 Bochar DA, Friesen JA, Stauffacher C & Rodwell VW (1999) Biosynthesis of mevalonic acid from acetyl-CoA In Comprehensive... reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis Proc Natl Acad Sci USA 95, 987 9–9 884 16 Kuzuyama T, Shimizu T, Takahashi S & Seto H (1998) Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis Tetrahedron Lett 39, 791 3–7 916 17 Jomaa... Lichtenthaler HK et al (1999) Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs Science 285, 157 3–1 576 18 Okuhara M, Kuroda Y, Goto T, Okamoto M, Terano H, Kohsaka M, Aoki H & Imanaka H (1980) Studies on new phosphonic acid antibiotics III Isolation and characterization of FR-31564, FR-32863 and FR-33289 J Antibiot (Tokyo) 33, 2 4–2 8 19 Kuemmerle HP, Murakawa T & De... Biosynthesis of terpenoids: 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Escherichia coli is a class B dehydrogenase FEBS Lett 465, 15 7– 160 31 Putra SR, Lois LM, Campos N, Boronat A & Rohmer M (1998) Incorporation of [2,3-13C2]- and [2,4-13C2]-D1-deoxyxylulose into ubiquinone of Escherichia coli via the mevalonate-independent pathway for isoprenoid biosynthesis Tetrahedron Lett 39, 2 3–2 6 32 Arigoni... by ultrafiltration (10 kDa cutoff) NADP+ (8.4 mg, 10 lmol), glucose dehydrogenase (57 lg, 12 units), d-glucose (75 mg) and IspC from E coli (800 lg) were added, and the pH was adjusted to 8.0 by the addition of 5 m NaOH (final volume, 11 mL) The reaction was controlled by 13C-NMR spectroscopy After 3 h, proteins were removed by ultrafiltration (10 kDa cutoff) The solution was lyophilized The residue was . Biosynthesis of isoprenoids – studies on the mechanism of 2C-methyl- D-erythritol-4-phosphate synthase Susan Lauw, Victoria Illarionova, Adelbert. The reductive reaction step has been shown to involve the transfer of a hydride ion from the pro-S position at C-4 of NADPH to the RE position of C-1 of