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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Protein Expr Purif. Author manuscript; available in PMC 2017 April 1.
Published in final edited form as:
PMCID: PMC4729580

Mechanistic Binding Insights for 1-Deoxy-D-Xylulose-5-Phosphate Synthase, the Enzyme Catalyzing the First Reaction of Isoprenoid Biosynthesis in the Malaria-Causing Protists, Plasmodium falciparum and Plasmodium vivax


We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both P. vivax and P. falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Keywords: 1-deoxy-D-xylulose-5-phosphate synthase, methylerythritol phosphate pathway, isoprenoids, malaria, Plasmodium vivax, Plasmodium falciparum

1. Introduction

Malaria is a life-threatening, infectious disease caused by parasites of the genus Plasmodium, transmitted through the bite of an infected female Anopheles mosquito. Malaria afflicts nearly 100 countries worldwide, with estimates of 198 million cases in 2013, resulting in 584,000 deaths. [1] Malaria poses a risk to roughly half of the population on earth, and infections are on the rise due to global warming, deforestation, and drug and insecticide resistance. [15] In humans, malaria is caused by an infection by one of five Plasmodium species: P. falicparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. P. falciparum malaria, constituting approximately 75% of the reported cases, is the most deadly form of malaria; however, P. vivax malaria, constituting approximately 20% of the reported cases, is serious and more widespread. [1, 6]

Isoprenoids (or terpenoids) constitute one of the largest classes of complex, biologically-active compounds, which serve diverse roles, functioning as cholesterol, hormones, fat-soluble hormones, carotenoids, pigments, and chlorophyll. [79] Isoprene is a five-carbon skeleton that is the precursor of all isoprenoids through rearrangement, cyclization, and oxidation. [10] Yet, isoprene itself is not used for the production of isoprenoids in vivo; instead the biological forms of isoprene are the two branched, phosphorylated five-carbon precursors, dimethylallyl diphosphate (DMAPP) and isopentyl diphosphate (IPP). For half a century, it was thought that the sole biosynthetic pathway of isoprenoid production was via the mevalonate pathway. [11] However, in 1996, Rohmer et al. [12] discovered that certain eubacteria, parasites, and several plants produce isoprenoids from a mevalonate-independent pathway. This biosynthetic route, known as the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, produces DMAPP and IPP from a set of orthogonal enzymes nonexistent in higher organisms and mammals. The MEP pathway, therefore, represents a platform for the development of novel herbicides, antimalarials, and antibiotics to aid the treatment of human disease. [1316]

In Plasmodium parasites, as well as other organisms from the phylum Apicomplexa, the MEP pathway produces DMAPP and IPP over seven successive enzymatic reactions. The first committed step of the pathway is catalyzed by 1-deoxy-D-xyluose-5-phosphate reductoisomerase (DXR), which involves the NADPH-dependent rearrangement and two electron reduction of the linear 1-deoxy-D-xylulose-5-phosphate (DXP) to the branched-chain isoprenoid precursor MEP. However, the enzyme catalyzing the first reaction of the pathway is also an attractive target for inhibitor development, 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS catalyzes the condensation of pyruvate and d-glyceraldehyde-3-phosphate (GAP) to produce CO2 and 1-deoxy-D-xylulose-5-phosphate (DXP), a reaction that is the rate-limiting for the MEP pathway in several organisms as well as the branch point for B vitamin and isoprenoid biosynthesis. [17] DXS belongs to the family of enzymes that utilizes thiamine diphosphate (ThDP) as a co-factor, and shares high homology with human transketolase, pyruvate dehydrogenase, and pyruvate decarboxylase. [1820] Like these other ThDP-dependent enzymes, DXS catalyzes decarboxylation and carboligation chemistry: a two-carbon intermediate bound to ThDP (hydroxyethyl-ThDP) is condensed with an acceptor substrate (GAP) via nucleophilic addition to generate a new carbon-carbon bond yielding DXP.

All ThDP-dependent enzymes catalyze two successive half reactions. [21] The first step involves the attack of an activated ThDP ylide on the first substrate. [22] The next step could occur via three distinct mechanisms. The most common is the classical ping-pong mechanism, in which the first product is released, forming a metastable enzyme-intermediate complex. Product formation follows after binding of the second substrate. Every ThDP-dependent enzyme studied to date follows this mechanism, and has also been proposed for DXS from E. coli and H. influenza. [23] Another possibility for the second half reaction proceeds through an ordered mechanism, where the first product can only be released after the second substrate is bound to the active site. The first substrate binds tightly and essentially irreversibly, while the second substrate can only bind to a pocket opened in the presence of the first substrate. Carbon dioxide (CO2) trapping studies on R. capsulatus DXS performed by Eubanks and Poulter suggested an ordered sequential kinetic mechanism. [24] The results of the experiment indicated that pyruvate bound first and irreversibly, followed by binding of GAP to form a ternary complex, and, finally, the release CO2 and DXP in that order. However, subsequent work demonstrating that DXS will catalyze the conversion of pyruvate to acetolatate and CO2 at high pyruvrate concentrations suggests that the kinetic mechanism for R. capsulatus DXS is not sequential. [25, 26]

The third potential mechanism that DXS could display is a random sequential mechanism, in which binding of both substrates is reversible and independent. Brammer et al. [25, 27] have proposed an unprecedented rapid equilibrium, random sequential mechanism for E. coli DXS, which is in contrast to all known ThDP-dependent enzymes to date. [28] Binding of either pyruvate or GAP is independent of the other, and GAP is required for the release of CO2 and the formation of a kinetically competent ternary structure. They suggest formation of the lactyl-ThDP intermediate is the overall rate-limiting step and it is maintained in a low-reactive state. GAP binding induces a reorientation to provide the optimal dihedral angle for decarboxylation. [29]

We report, herein, on the successful production of recombinant, catalytically active forms of Plasmodium falciparum DXS and Plasmodium vivax DXR. Similar to earlier work on the expression of P. vivax DXS [30] and P. falciparum DXR, [31] the catalytically active forms of P. falciparum DXS and P. vivax DXR have been truncated to their respective catalytic cores, lacking the signaling and transit peptide for each of these enzymes. In addition to recombinant P. falciparum DXS and P. vivax DXR, we also produced the truncated forms of P. vivax DXS and P. falciparum DXR in E. coli by following published protocols. Both P. falciparum and P. vivax DXS enzymes were characterized via steady-state kinetic analyses, intrinsic tryptophan fluorescence titrations, and dead-end inhibition studies employing a pair of pyruvate mimics. DXS activity was measured using a DXR-dependent coupled assay, pairing P. falciparum DXS with P. falciparum DXR or P. vivax DXS with P. vivax DXR. Our characterization of DXS from two Plasmodium species points toward a random sequential kinetic mechanism, an unusual finding for ThDP-dependent enzymes. This work fosters a deeper understanding of the DXS-catalyzed reaction and will aid in the rational design of DXS inhibitors for the treatment of malaria and other human diseases.

2. Experimental Procedures

2.1. Materials and General Methods

Unless otherwise noted, all reagents were obtained from commercial sources. BL21 (DE3) E. coli cells and the pET-28a (+) vector were purchased from Novagen. PfuUltra High-Fidelity DNA polymerase was purchased from Aglient. BamHI, XhoI, NdeI, Antarctic Phosphatase, and T4 DNA ligase were purchased from New England Biolabs. Kanamycin monosulfate and IPTG were purchased from Gold Biotechnology. Size exclusion chromatography was performed on a GE Healthcare AKTAprime Plus FPLC, coupled to a HiLoad 16/600 Superdex 200 pg column. Spectrophotometric analyses were performed on a Cary 300 Bio UV-Visibile spectrophotometer. Tryptophan fluorescence studies were performed on a JASCO FP-8300 spectrofluorometer.

2.2. Cloning of the P. falciparum dxs and P. vivax dxr Genes Without Signal and Transit Peptides

A synthetic, codon-optimized gene for P. falciparum dxs was purchased from Genscript. P. falciparum dxs was designed with 5’-NdeI and 3’-XhoI restriction sites and synthesized into a pUC57 vector. The full-length gene was excised from the pUC57 vector and cloned into the NdeI and XhoI restriction sites of the pET-28a vector. Similarly, a P. vivax dxr codon-optimized gene was also purchased from Genscript, with 5’-NdeI and 3’-BamHI restriction sites in a pUC57 vector. P. vivax dxr was cloned into pET-28a using NdeI and BamHI restriction sites.

Truncated gene constructs were produced from the full length, codon-optimized genes using PCR extraction to eliminate expression of the N-terminal signal and transit peptides of both PfDXS and PvDXR. PfDXS-A, PfDXS-B, PfDXS-C, and PfDXS-D were produced with deletions of 237, 286, 292, and 310 amino acids, respectively. PvDXR-E, PvDXR-F, and PvDXR-G were constructed with 99, 106, and 110 amino acid deletions, respectively. The cloning of PvDXS was described previously. [30] The cloning of PfDXR has also been previously described, [31] and this gene was generously donated by Dr. Nobutada Tanaka at Showa University (Japan) for the use in this study.

2.3. Overexpression of Truncated DXS and DXR Proteins in E. coli

Truncated gene transcripts of DXS and DXR in pET-28a were transformed into E. coli BL-21 (DE3) cells. A fresh colony of cells harboring either truncated Pfdxs-pET-28a or Pvdxr-pET-28a were inoculated at 37 °C in LB broth containing 40 µg/mL kanamycin supplemented with 0.8% glucose and 25 mM potassium phosphate (pH = 7.2). The cultures were grown until the OD600 reached 0.3, then diluted 1:100. For the overexpression of PfDXS, cells were cultured until the OD600 reached 0.6, the temperature reduced to 10 °C, induced by the addition of 0.5 mM IPTG, and incubated overnight (12 hours) with continuous shaking. To produce PvDXR, the temperature was dropped to 30 °C after achieving an OD600 of 0.6, induction initiated with 1 mM IPTG, and overnight incubation. Cells were harvested by centrifugation, and the cell pellets stored at −80 °C. The expression of PvDXS [30] and PfDXR [31] were described previously.

2.4. Purification of DXS and DXR by Ni-NTA Affinity and Size-Exclusion Chromatography

Cells were thawed on ice and all purification steps were performed at 4 °C. Cell pellets (4 g) were resuspended in 20 mL of binding buffer supplemented with protease inhibitors: 20 mM Tris pH 7.5, 500 mM NaCl, 20 mM imidazole, 10 mM b-mercaptoethanol (BME), 1 mM phenylmethylsulfonyl fluoride (PMSF), 4 mg/mL leupeptin, 2 mg/mL pepstatin, and 43 mg of an E. coli-specific protease inhibitor cocktail (Sigma P8465). The cells were lysed via sonication, centrifuged at 16,000 × g for 20 minutes at 4 °C, and the recombinant enzyme-containing soluble lysate was retained.

Purification of DXS and DXR was facilitated by the fusion of an N-terminal His6-tag to the active, truncated enzymes. The supernatant was applied to a borosilicate glass column (1.5 cm × 5 cm) containing 5 mL of nickel-nitrilotriacetic acid (Ni-NTA) resin equilibrated with binding buffer. The non-bound proteins were first eluted with 5 column volumes (CVs) of binding buffer, then with 20 CVs of wash buffer (20 mM Tris pH 7.5, 500 mM NaCl, 60 mM imidazole, and 10 mM BME) at a flow rate of 1 mL/minute. Tagged, recombinant enzyme was eluted with 2 CVs of elution buffer (20 mM Tris pH 7.5, 500 mM NaCl, 250 mM imidazole, 10 mM BME) and concentrated to <3 mL using an Amicon stirred ultrafiltration cell in preparation for size exclusion chromatography (SEC). The concentrated sample was injected onto an AKTAprime FPLC coupled to a 16/600 Superdex column equilibrated with 50 mM Tris pH 7.5, 150 mM NaCl, and 10 mM BME. The flow rate for the SEC was set to 1 mL/min with a maximum pressure of 0.5 mPa. The concentration of the enzyme-containing fractions was determined using the Bradford assay and purity was assessed by a 10% SDS-PAGE gel visualized with Coomassie Brilliant Blue stain. Fractions containing purified enzyme were combined, glycerol added to a final concentration of 10% (w/v), and the aliquots were flash frozen in liquid nitrogen with subsequent storage at −80 °C.

2.5. Kinetic Analyses

DXS activity from both P. falciparum and P. vivax was measured spectrophotometrically using DXR from its corresponding organism as a coupling enzyme as previously reported [32] with minor variations. The concentration of D-GAP was determined enzymatically from commercially available D,L-GAP using human GAPDH. [26] Throughout the manuscript, our use of “GAP” refers to the concentration of D-GAP in the D,L-GAP mixture. Reaction mixtures containing 100 mM HEPES pH 7.0, 100 mM NaCl, 1.5 mM MgCl2, 1 mg/mL BSA, 1 mM ThDP, 2 mM BME, 150 µM NADPH, GAP, and pyruvate were preincubated for 5 minutes at 37 °C. DXR was pipetted into the cuvette (0.2 mg/mL), 100 nM of DXS was added to initiate the reaction, and the solution (750 µL total volume) was vigorously mixed before initializing the spectrophotomer software. Rates of NADPH consumption in the coupled step between DXS and DXR were monitored spectrophotometrically at 340 nm for 2 minutes (extinction coefficient of 6,220 M−1 cm−1 for NADPH). The rates of NADPH depletion were used to calculate initial rates of DXP formation. The apparent kinetic constants were determined by fitting the resulting data to equation 1 using SigmaPlot 12.0: vo represents initial velocity, Vm,app is the apparent maximal velocity, Km,app is the apparent Michaelis constant, and [S] is the substrate concentration. Assays were performed in triplicate and the uncertainty for the kcat,app and (kcat/Km)app values were calculated using equation 2, where σ is the standard error. [33]

Equation 1

Equation 2

To delineate between a sequential and classic ping-pong mechanism, double-reciprocal plots of the initial velocity data were generated for GAP and pyruvate in SigmaPlot 12.0. Non-linear regression analysis of initial rates and model discrimination analyses were performed in WaveMetrics IGOR Pro 6. Initial velocities were determined by varying the concentration of one substrate, while holding the other substrate at a fixed concentration. GAP was evaluated between 3.5 µM and 16 µM, whereas pyruvate was tested at concentrations ranging from 50 µM to 250 µM. The resulting initial velocity data was fit to equation 3 for a rapid-equilibrium random Bi-Bi mechanism, where vo is the initial velocity, Vm is the maximal velocity, [A] is the concentration of substrate A, [B] is the concentration of substrate B, Kia is the dissociation constant for substrate A, Kb is the Michaelis constant for substrate B, and Ka is the Michaelis constant for substrate A.

Equation 3

2.6. Synthesis of Methylacetylphosphonate (MAP)

MAP was synthesized, as previously reported [25, 3436], with minor variations. In a dry three-necked round bottom flask with a magnetic stir bar under positive N2 pressure, acetyl chloride (1.21 mL, 14 mmol, 1 eq) was cooled to 0 °C in an ice-water bath. Trimethyl phosphite (1.65 mL, 14 mmol, 1 eq) was slowly injected over a period of 3 hours, which resulted in evolution of MeCl. After full addition of trimethyl phosphite, the light-grey solution was allowed to warm to room temperature and stirred for an additional 8 hours. Unreacted reagents were removed under vacuum to afford acetylphosphonate dimethyl ester (DAP). 1.58 g recovered (10.4 mmol, 74% yield) as a pale grey oil. 1H NMR (400 MHz, CDCl3) δ 2.24 (d, J = 5.31 Hz, 3H, CH3), 3.61 (d, J = 10.69, 6H, OCH3). 13C NMR (400 MHz, CDCl3): d= 208.1 (d, JP,C=169.8 Hz), 54.0 (d, 2JP,C=7.2 Hz, 2 C), 20.9 (d, 2JP,C=36.0 Hz).

The recovered DAP (1.58 g, 10.4 mmol, 1 eq) was dissolved in dry acetone (10.4 mL), and the solution was slowly injected into a solution of sodium iodide (1.71 g, 11.5 mmol, 1.1 eq) in 6 mL dry acetone. The yellow reaction mixture was stirred overnight at room temperature after which the precipitate was filtered, triturated with dry acetone, and dried to yield purified MAP. 93% yield. 1H NMR (400 MHz, D20) δ 2.20 (3 H, d, J 9.8 Hz) and 3.69 (3 H, d, J 10.63 Hz). All NMR spectra of isolated compounds are in agreement with literature reports.

2.7. Inhibition of DXS with β-Fluorpyruvate and Methylacetylphosphonate

The varying inhibition patterns of DXS activity were measured with the DXS-DXR coupled assay in the presence of either β-fluorpyruvate or methylacetylphosphonate (MAP). Inhibitor concentrations were based upon previously established IC50 values (data not shown) and added to the kinetic reaction mixtures described above. For PvDXS, the concentrations of β-fluorpyruvate tested were 0, 35, and 75 µM, while MAP was analyzed at 0, 150, and 300 µM. To test PfDXS inhibition, β-fluorpyruvate and MAP concentrations were held at 0, 50, 100 µM and 0, 70, 140 µM, respectively. No inhibition of DXR by either inhibitor was observed. All assays were performed in triplicate, and the resulting data was fit to equations 46 in SigmaPlot12.0 for competitive, noncompetitive, and uncompetitive inhibition, respectively. For equations 46, vo is the initial velocity, Vm,app is the apparent maximal velocity, Km,app is the apparent Michaelis constant, [S] is the substrate concentration, [I] is the inhibitor concentration, and Ki is the inhibition constant.

Equation 4

Equation 5

Equation 6

2.8. Intrinsic Tryptophan Fluorescence Measurements for the Determination of Binding Constants for DXS

To study the binding of pyruvate and GAP to DXS, intrinsic tryptophan fluorescence (ITF) quenching titrations were utilized. All reaction mixtures for ITF titrations contained 100 mM HEPES pH 7.0, 100 mM NaCl, 1.5 mM MgCl2, 2 mM BME, and 500 nM DXS in a final volume of 400 µL. Fluorescent emission spectra were acquired on a JASCO FP-8300 spectrofluorometer at 25 °C. The excitation wavelength was set at 280 nm, and emission occurred between 290–300 nm. Spectra were measured in triplicate from 305 nm to 360 nm, using a scan speed of 50 nm/min, and both excitation and emission bandwidth set at 2.5 nm. Spectra were corrected by subtracting emission spectra from acquisitions containing no enzyme (blanks), and normalized by taking the ratio of the fluorescence emission intensity at the λmax relative to starting enzyme only samples. Data were fit to equation 7, where ΔF is the change in intrinsic fluorescence, ΔFmax is the maximum change of fluorescence at infinite ligand concentration, [L] is the ligand concentration, and Kd is the ligand dissociation constant.

Equation 7

3. Results and Discussion

Malaria parasites and other infectious organisms of the phylum Apicomplexa possess the apicoplast, an endosymbiotic organelle similar to the chloroplast of plants and algae. [37] With a complete genome producing nearly 400 assorted proteins, the apicoplast is vital in various aspects of parasite metabolism including fatty acid and isoprenoid biosynthesis. All of the enzymes from the MEP pathway are nuclear-encoded and translocated to the apicoplast through a secretory pathway utilizing a bipartite N-terminal signaling motif. This sequence consists of two functional domains: an endoplasmic reticulum-targeting signal peptide and a transit peptide. The signal peptide directs the translocation of the enzyme into the secretory pathway, while the transit peptide facilitates the movement through the four outer membranes of the apicoplast. Once inside, the peptides are cleaved off (most likely by a stromal processing protease) to yield the catalytically-competent enzyme. [38]

To date, only a few enzymes targeted to the apicoplast from Plasmodium have been expressed, due in part to difficulties in obtaining soluble recombinant enzyme from E. coli host cells. [30, 39, 40] In addition to codon biases between E. coli and Plasmodium species (high GC content, ribosomal binding sites, random repeats of rare codons, etc), the presence of the signaling and transit motifs consistently result in the accumulation of insoluble inclusion bodies. [39] Therefore, serial deletion of the full-length, untruncated gene is required to produce a DXS or DXR variant that is soluble when expressed in E. coli. Several freely available algorithms (PlasmoAP [41], PATS [42], PSORT [43], etc.) have been developed, which have proven successful in identifying the signal peptide. Note that the signal peptide varies in length from 20–30 amino acids and exhibits a consensus sequence. In contrast, there is no consensus sequence or narrowly defined size for the transit peptide, which can vary from 50–500 amino acids, meaning that there are no current algorithms that can accurately predict this peptide region (see Figure 1 of the DXS amino acid sequence from several organisms with low N-terminal homology). [20] Malarial transit peptides can be approximated by the presence of highly enriched lysine and asparagine residues, which results in an overall positive charge. [41] This trend is pronounced in the amino acid sequence of P. falciparum DXS between residues ~150–300 (Figure 1, note the high number of successive asparagine residues).

Figure 1
Homology overlays of the N-terminal amino acid sequence for Plasmodium vivax, Plasmodium knowlesi, Plasmodium cynomolgi and Plasmodium falciparum.

By utilizing homology modeling and N-terminal serial deletions of the full-length codon-optimized genes, several targeted variants of PfDXS and PvDXR were produced in our expression optimization study. PfDXS-A, PfDXS-B, PfDXS-C, and PfDXS-D were generated with N-terminal deletions of 237, 286, 292, and 310 amino acids, respectively. PvDXR-E, PvDXR-F, and PvDXR-G were constructed with N-terminal deletions of 99, 106, and 110 amino acid deletions, respectively. Each of these genes was then transformed into BL-21 (DE3) E. coli expression cells, and cultured in 2 mL aliquots. These small-scale cultures were optimized for time of expression (1, 3, 5, and 12 hours), induction temperature (10, 25, 30, or 37 °C) and IPTG concentration (0.1, 0.5, or 1.0 mM) for a total of 48 culture conditions per truncated gene. The small scale cultures were then centrifuged, lysed via sonication, and the soluble proteome subjected to an anti-His6 Western blot antibody assay for the detection of recombinant enzyme. Only one truncated gene and one set of variables proved successful for each enzyme: PfDXS-D (with 310 amino acids deleted) was expressed at 10 °C, induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and incubated overnight (12 hours) with continuous shaking. After subsequent purification via Ni-NTA affinity and size exclusion chromatography, the final yield of PfDXS-D was 1.4 mg/L (Figure 2). To produce PvDXR, the protein construct with 110 amino acids removed (PvDXR-G) produced soluble enzyme at 30 °C with 1 mM IPTG and overnight incubation. PvDXR-G yielded 0.9 mg/L after both affinity and size exclusion chromatography (Figure 3). This enzyme was used in the DXS-DXR coupled assay to further study PvDXS (kinetic constants of PvDXR-G: Km = 48 ± 1.6 µM, Vm = 1.3 ± 0.01 s−1, kcat/Km = (2.7 ± 0.1) × 104 M−1s−1. These values are in good agreement with previously published data for DXR from several other organisms. [4447] The cloning, expression, and characterization of PfDXR [31] and PvDXS [30] were previously described. From this point forth, PfDXS-D and PvDXR-G will simply be referred to as PfDXS and PvDXR, respectively.

Figure 2
Purification scheme of Plasmodium falciparum DXS.
Figure 3
Purification scheme of Plasmodium vivax DXR.

Once both sets of enzymes were cloned, expressed, and purified, we set about to elucidate kinetic and binding characteristics of DXS from P. falciparum and P. vivax. For the determination of kinetic constants, initial velocities of DXP formation were monitored using the DXS-DXR coupled assay at sub-saturating concentrations of one substrate, while holding the other fixed. Rates were determined in triplicate. Binding constants were obtained using intrinsic tryptophan fluorescence titrations, and the results of both of these experiments are detailed in Table 1.

Table 1
Binding Constants and Steady-State Kinetic Constants of DXS from several organisms

The kinetic constants for both P. falciparum DXS and P. vivax DXS are comparable to previously published data for DXS from E. coli, R. capsulatas, and D. radiodurans. The Kd values for both Plasmodium enzymes are also commensurate with the dissociation constants for E. coli DXS (see Figures 4 and and5).5). Clues of the binding mechanism can be delineated from these data: in the ordered mechanism with pyruvate binding first and, irreversibly, binding of pyruvate causes a conformational change which promotes binding of GAP. Binding of GAP to free enzyme should, therefore, be negligible. [25] However, a measurable binding constant for pyruvate to both PfDXS and PvDXS suggests that pyruvate binding is, in fact reversible, inconsistent with an ordered mechanism. In the proposed ping-pong mechanism, GAP will have low affinity for the free enzyme. However, again, we observed quenching of the fluorescent signal indicative of GAP binding reversibly to both of the Plasmodium DXS enzymes. These data are inconsistent with a ping-pong mechanism. Taken together, the intrinsic fluorescence data supports a random sequential mechanism where GAP and pyruvate can both bind reversibly and independently to either P. falciparum or P. vivax DXS.

Figure 4
Substrate-binding analysis of PvDXS by intrinsic-tryptophan quenching titrations with increasing [pyruvate] and [GAP].
Figure 5
Substrate-binding analysis of PfDXS by intrinsic-tryptophan quenching titrations with increasing [pyruvate] and [GAP].

Also noted in our ITF data is an apparent Stokes shift in the wavelength for maximum fluorescence intensity when DXS is incubated with high concentrations of GAP, which was not found at similarly high levels of pyruvate (Figure 4). Tryptophan fluorescence is commonly used to monitor perturbations in protein dynamics and localized structures; fluorescence from tryptophan is sensitive to the polarity of its local environment. [48] Therefore, this apparent Stokes shift suggests that GAP binding induces a conformation change in DXS, at least within the vicinity of the fluorescent tryptophan residue. This represents the first report of a conformational change in DXS upon substrate binding. Additional data for other DXS enzymes is required to determine the significance of our observation.

Double-reciprocal plots were generated to differentiate between a sequential binding mechanism and a ping-pong mechanism for both of the DXS enzymes from Plasmodium (Figures 6 and and7).7). The Lineweaver-Burk plots for both GAP and pyruvate had noncompetitive pattern with changes in both slope and intercept, suggesting a random sequential mechanism. If substrate binding to DXS were ordered, a competitive pattern for GAP would result and an uncompetitive pattern for pyruvate would be observed. Substrate inhibition was found at higher concentrations of both pyruvate and GAP (as evidenced by the converging lines), similar to what was reported by Brammer, et al. [25] for E. coli DXS. The data was further evaluated using global non-linear regression analysis: several mechanistic models for a two substrate, two product (Bi Bi) system were examined in a model discrimination analysis (rapid-equilibrium ordered, rapid-equilibrium random, ping-pong, etc). The data were best fit to the equations for a random sequential mechanism (Figures 8 and and99).

Figure 6
PvDXS double-reciprocal plots of initial velocities for pyruvate and GAP. (A) Velocities measured at fixed concentrations of GAP: 0.016 mM ([large star]), 0.008 mM (●), 0.005 mM (■), 0.0035 mM (▲). (B) Velocities measured at fixed ...
Figure 7
PfDXS double-reciprocal plots of initial velocities for pyruvate and GAP. (A) Velocities measured at fixed concentrations of GAP: 0.016 mM ([large star]), 0.008 mM (●), 0.005 mM (■), 0.0035 mM (▲). (B) Velocities measured at fixed ...
Figure 8
PvDXS global non-linear regression analysis for a random sequential mechanism. (A) Velocities measured at fixed concentrations of GAP: 0.016 mM ([big up triangle, open]), 0.008 mM (◊), 0.005 mM (□), 0.0035 mM (○). (B) Velocities measured at ...
Figure 9
PfDXS global non-linear regression analysis for a random sequential mechanism. (A) Velocities measured at fixed concentrations of GAP: 0.016 mM ([big up triangle, open]), 0.008 mM (◊), 0.005 mM (□), 0.0035 mM (○). (B) Velocities measured at ...

Finally, inhibition studies were conducted to further confirm the mechanism of substrate binding to DXS. Both β-fluoropyruvate and MAP have been previously used as pyruvate mimics to study ThDP-dependent enzyme mechanisms. [34, 4951] After ThDP activation, β-fluoropyruvate undergoes a catalytic step to release CO2 with consequent elimination of fluoride; therefore, it is not representative of a true dead-end inhibitor against pyruvate. MAP cannot undergo decaboxylation, and is, accordingly, a more suitable pyruvate analog. [25] Initial rates of DXP production were monitored using the DXS-DXR coupled assay at sub-saturating, varied concentrations of one substrate at fixed concentrations of the second substrate, in the presence of either inhibitor at varied concentrations. The equations for competitive, noncompetitive, and uncompetitive inhibition were fit to the data, and the results of the best fits are shown in Figures 10 and and11.11. For the pair of DXS enzymes, both MAP and β-fluoropyruvate displayed competitive inhibition against pyruvate, while exhibiting noncompetitive inhibition versus GAP (Table 2). This pattern of results is most consistent with a random sequential binding mechanism for both substrates to DXS. Conversely, a ping-pong mechanism would yield an uncompetitive pattern with respect to GAP.

Table 2
Inhibition constants for β-fluoropyruvate and MAP against PvDXS and PfDXS

4. Conclusion

The first two enzymes of the MEP pathway in the malaria-causing protists Plasmodium vivax and Plasmodium falciparum are DXS and DXR, respectively. Catalytically active, truncated forms of all four enzymes have been cloned and over-expressed in E. coli: P. falciparum DXS (described herein), P. falciparum DXR by Umeda et al.26, P. vivax DXS by Handa et al.25, and P. vivax DXR (described herein). Because of the metabolic centrality of the isoprenoids, the availability of DXS and DXR from P. falciparum and P. vivax should facilitate the development of novel anti-malarials targeted against either of these enzymes. The kinetic mechanism for DXS has proven controversial, with the majority of the published data pointing towards a random sequential mechanism. [25, 27, 28] Such a mechanism is unusual for a ThDP-dependent enzyme, as the first substrate typically forms an adduct with the thiazolium moiety of ThDP. [21, 22, 27] yielding either an ordered or ping-pong kinetic mechanism. Our characterization of PfDXS and PvDXS, steady-state kinetic analyses, substrate binding studies, and dead-end inhibition experiments, are most consistent with a random sequential mechanism for both enzymes. The expression of PfDXS and PvDXS in E. coli, providing straightforward access to multi-milligram amounts of protein and fostering the construction of site-directed mutants, will enable the detailed structure-function studies to better understand the unique aspects of ThDP-dependent chemistry of DXS.

Figure 10
Inhibition of DXS by β-fluorpyruvate. (A) PvDXS assayed with varied concentrations of pyruvate at fixed concentrations of GAP (26 µM), in the presence of β-fluorpyruvate: 0 µM (●), 35 µM (○), and ...
Figure 11
Inhibition of DXS by MAP. (A) PvDXS assayed with varied concentrations of pyruvate at fixed concentrations of GAP (26 µM), in the presence of MAP: 0 µM (●), 150 µM (○), and 300 µM ([triangle]). (B) PvDXS ...


  • The novel truncation and expression of Plasmodium falciparum DXS and Plasmodium vivax DXR.
  • Enzymatic characterization by steady-state kinetics, inhibition, and binding constants.
  • Confirmed previous reports of a random sequential binding mechanism for both Plasmodium falciparum and Plasmodium vivax DXS.


The authors would like to thank Dr. Nobutada Tanaka at Showa University (Japan) for donating the Plasmodium falciparum dxr gene, as well as Dr. Daniel R. Dempsey (Johns Hopkins University) for helpful discussions throughout the completion of this study.

Funding Disclosure

This research was supported in part by grants from the Florida Center of Excellence for Biomolecular Identification and Targeted (FCoE-BITT), the Shirley W. and William L. Griffin Foundation, National Institute of Drug Abuse at the National Institutes of Health (R03-DA034323), and National Institute of General Medical Science of the National Institutes of Health (R15-GM107864) to D.J.M.


1-deoxy-D-xylulose-5-phosphate synthase
1-deoxy-D-xyluose-5-phosphate reductoisomerase
isopentyl diphosphate
isopropyl β-D-1-thiogalactopyranoside
dimethylallyl diphosphate
2-C-methyl-D-erythritol 4-phosphate
thiamine diphosphate


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