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Molecular & Biochemical Parasitology 137 (2004) 43–53

Molecular characterization of bifunctional hydroxymethyldihydropterin

pyrophosphokinase-dihydropteroate synthase from

Plasmodium falciparum

Waraporn Kasekarn a , Rachada Sirawaraporn a , Thippayarat Chahomchuen a ,

Alan F. Cowman b , Worachart Sirawaraporn a,c,∗

a Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

b The Walter and Elisa Hall Institute of Medical Research, 1G Royal Parade, Parkville, Vic. 3050, Australia

c Center for Vector and Vector-Borne Diseases, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

Received 20 November 2003; received in revised form 1 April 2004; accepted 9 April 2004

Available online 26 May 2004

Abstract

A 2118-base pair gene encoding the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate syntheses of Plasmodium

falciparum (pfPPPK-DHPS) was expressed under the control of the T5 promoter in a DHPS-deficient Escherichia coli strain. The

enzyme was purified to near homogeneity using nickel affinity chromatography followed by gel filtration and migrates as an intense band

on sodium dodecyl sulfate–polyacrylamide gel electrophoresis with apparent mass of ∼83 kDa. Gel filtration suggested that the native

pfPPPK-DHPS might exist as a tetramer of identical subunits. The enzyme was found to be Mg 2+ - and ATP-dependent and had optimal

temperature ranging from 37 to 45 ◦ C with peak activity at pH 10. Sodium chloride and potassium chloride at 0.2 and 0.4 M, respectively,

activated the activity of the enzyme but higher salt concentrations were inhibitory. Guanidine–HCl and urea inhibited the enzyme activity

by 50% at 0.25 and 0.9 M, respectively. Kinetic properties of the recombinant pfPPPK-DHPS were investigated. Sulfathiazole and dapsone

were potent inhibitors of pfPPPK-DHPS, whilst sulfadoxine, sulfanilamide, sulfacetamide and p-aminosalicylic acid were less inhibitory.

Our construct provides an abundant source of recombinant pfPPPK-DHPS for crystallization and drug screening.

© 2004 Elsevier B.V. All rights reserved.

Keywords: Malaria; Plasmodium falciparum; Hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate syntheses; Sulfa drug target

1. Introduction

The parasite Plasmodium falciparum is responsible for

hundreds of millions of cases and kills approximately 2.7

millions people each year [1]. Resistance of P. falciparum

to currently available antimalarials has become widespread

and highlights the need for new potent antimalarial agents.

Several enzymes in the folate metabolic pathway are crucial

for the growth of the malaria parasite, some of which

are absent in the human host and are therefore potential

Abbreviations: pfPPPK-DHPS, Plasmodium falciparum hydroxymethyldihydropterin

pyrophosphokinase-dihydropteroate syntheses;

pfDHFR-TS, Plasmodium falciparum dihydrofolate reductase-thymidylate

syntheses; pABA, p-aminobenzoic acid; PAS, p-aminosalicylic acid; Ni-

NTA, nickel nitriloacetic acid; IPTG, isopropyl--d-thiogalactopyranoside;

MM, minimal media; DMSO, dimethyl sulfoxide; PCR, polymerase

chain reaction

∗ Corresponding author. Tel.: +66 2 201 5605; fax: +66 2 248 0375.

E-mail address: scwsr@mahidol.ac.th (W. Sirawaraporn).

targets for malarial chemotherapy. Hydroxymethyldihydropterin

pyrophosphokinase-dihydropteroate syntheses

(pfPPPK-DHPS) of P. falciparum is a target of sulfadoxine

[2]. Formulated as a fixed-dose combination with antifolate

inhibitors such as pyrimethamine [3], the combination

(Fansidar ® ) has been widely exploited for the treatment of

uncomplicated “falciparum” malaria in Africa where these

parasites are resistant to chloroquine [4].

Hydroxymethyldihydropterin pyrophosphokinase (PPPK;

EC 2.7.6.3) catalyzes the ATP-dependent pyrophosphorylation

of 6-hydroxymethyl-7,8-dihydropterin (H 2 PtCH 2 OH)

to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate.

The latter is then condensed with p-aminobenzoic acid

(pABA) to yield 7,8-dihydropteroate, a crucial precursor

in the subsequent reaction catalyzed by dihydropteroate

syntheses (DHPS; EC 2.5.1.15) in the biosynthesis of

tetrahydrofolate [5]. The sulfonamide and sulfone group

of compounds have been used extensively in the treatment

of bacterial diseases. These inhibitors compete with the

0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.molbiopara.2004.04.012


44 W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53

natural substrate, pABA, for binding to the enzyme [6],

causing depletion of dihydrofolate (H 2 folate) and inhibition

of parasite growth. The gene encoding DHPS from a number

of organisms have been reported; these include dhps of

Streptococcus pneumoniae [7,8], Bacillus subtilis [9], Escherichia

coli [10], Neisseria meningitidis [11], Mycobacterium

tuberculosis [12], and Mycobacterium leprae [12].

Unlike DHPS of prokaryotes, the enzyme of protozoan parasites

such as P. falciparum [13–15] and Toxoplasma gondii

[16] is a bifunctional enzyme with PPPK. In Pneumocystis

carinii, however, the DHPS is a trifunctional enzyme with

dihydroneopterin aldolase (DHNA) and PPPK [17]. Thus

far, crystal structures from only three bacterial DHPSs have

been solved [18–20].

The decreased susceptibility of malaria parasites to antifolates

has been a major problem for the control of malaria.

Previous analyses of the dhps gene of antifolate-resistant P.

falciparum strains from a wide range of geographic areas

have shown that the mechanism of sulfadoxine resistance involves

point mutations in the DHPS domain [13,15,21–23].

A thorough understanding of the molecular basis of drug

resistance is therefore necessary for further improvement

of the effectiveness of the inhibitors to combat resistant

parasites. Our goal is to employ a structure-based approach

to understand the molecular basis of sulfadoxine resistance

and to develop a simple system which can produce sufficient

enzyme for structural studies and screening of novel

effective inhibitors against pfDHPS. Thus far, the lack of

sufficient enzyme has precluded structural studies of this

chemotherapeutically important target. Although expression

of pfPPPK-DHPS has previously been reported [15], the

amount of enzyme produced was insufficient for structural

studies. In this paper, we describe an expression system

which produces sufficient amounts of highly active pfPPPK-

DHPS. The enzyme was purified, characterized and the

kinetic parameters for its substrates were determined and

compared to previously published results. An extensive series

of inhibition studies by sulfa drugs was carried out, and

the relative inhibitory activities of sulfa drugs were compared.

The availability of ample amounts of recombinant

enzyme resulting from this work will not only facilitate the

basic molecular approaches and development of medium- to

high-throughput drug screening, but also provide a unique

source of this chemotherapeutically important enzyme for

the development of novel selective antimalarials.

2. Experimental procedures

2.1. Materials

Restriction endonucleases and other DNA-modifying

enzymes were obtained from New England Biolabs and

Promega. Plasmid DNA extraction, purification kits and

Ni-NTA resin were from QIAGEN. Radioactive [carboxyl-

14 C] pABA (specific activity 58 mCi mmol −1 ) was from

Moravek Biochemicals. 6-Hydroxymethylpterin, sulfathiazole,

sulfapyridine, sulfadimethoxine, sulfacetamide, sulfanilamide

and p-aminosalicylic acid were purchased from

Sigma. Other sulfonamides used in the study were either

from ICN Biomedical or from Aldrich. Sulfadoxine was

a gift from D. Jacobus (Jacobus Pharmaceutical Co., Inc).

All other chemicals and reagents were of the highest purity

commercially available.

2.2. Plasmids and bacterial strains

A full-length pppk-dhps gene of P. falciparum (modified

slightly to improve gene stability) in the vector pTrc

[15] was used as a template for subcloning into pKOS007-

90-PL expression vector [12]. Escherichia coli XL1-Blue,

employed as a general host strain for plasmid manipulation,

was from Stratagene. A DHPS-deficient strain E. coli

C600folP::Km r [11] was used as a host strain for the expression

of pfPPPK-DHPS.

2.3. Construction of expression plasmids

The gene encoding the wild-type pfPPPK-DHPS in pTrc

vector [15] was amplified using the oligonucleotide primers

pfPPPK-DHPS-1 (5 ′ -GCAGGCCATATGGAAACTATACA-

AG-3 ′ ) and pfPPPK-DHPS-2 (5 ′ -CGCGGATCCCATGTTT-

GCACTTTCC-3 ′ ). The NdeI and BamHI recognition sites,

shown underlined in the primers pfPPPK-DHPS-1 and

pfPPPK-DHPS-2, respectively, were introduced to facilitate

cloning of the amplified product into the expression vector.

The amplified DNA was digested with NdeI and BamHI,

gel-purified, and ligated with pKOS007-90PL plasmid

predigested with NdeI and BamHI. The resulting recombinant

plasmid, pKOS-pfPPPK-DHPS, was used as a template

for the construction of pKOS-pfPPPK-DHPS(His) using

the primers pfPPPK-DHPS-1 and pfPPPK-DHPS-3 (5 ′ -

CATAAGCTTAGTGGTGGTGGTGGTGGTGCACTTGG-

TCTATTTT-3 ′ ). The PCR reaction (100 l) in 1× PCR

buffer (Promega) consisted of 0.1 g ofpfpppk-dhps DNA

template, 20 pmol each of the primers, 200 M each of

dNTPs, 1.5 mM MgCl 2 and 2.5 U of Taq DNA polymerase.

The reaction was performed for 25 cycles; each

cycle consisted of 94 ◦ C for 45 s, 42 ◦ C for 30 s, and

72 ◦ C for 60 s. The amplified DNA (∼2.1 kb) was gelpurified,

ligated into pGEM-T Easy vector and transformed

into E. coli XL1-Blue. The recombinant plasmid was digested

with NdeI/HindIII to excise the pfpppk-dhps fragment

for subsequent cloning into the corresponding sites

of pKOS007-90PL vector. The resulting clone, pKOSpfPPPK-DHPS(His),

was verified for its sequence and was

transformed into E. coli C600folP::Km r for expression

experiments.

2.4. Expression and purification of pfPPPK-DHPS

Fresh overnight culture from a single colony of E. coli

C600folP::Km r harboring pKOS-pfPPPK-DHPS(His) was


W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53 45

inoculated at 1% inoculum into Luria-Bertani (LB) media

supplemented with 100 gml −1 ampicillin and 40 gml −1

kanamycin. The culture was grown at 37 ◦ C with shaking

and isopropyl--d-thiogalactopyranoside (IPTG) at a

final concentration of 1 mM was added when A 600 was

∼0.5–0.6. After continuous shaking at 30 ◦ C for 12 h,

the cells were pelleted by centrifugation at 10,000 × g

for 10 min at 4 ◦ C and were resuspended in lysis buffer

(50 mM NaH 2 PO 4 , pH 8.0, 500 mM NaCl, 10 mM imidazole,

20 mM -mercaptoethanol and 10% glycerol) containing

10 gml −1 leupeptin, 20 gml −1 phenylmethylsulfonyl

fluoride, 20 gml −1 trypsin inhibitor, 20 gml −1

aprotinin and 1 mM benzamidine–HCl. The cells were disrupted

twice by French Press at 15,000 psi. After removing

the cell debris by centrifugation at 30,000 × g for 1 h at

4 ◦ C, the clear supernatant was applied to a Ni-NTA column

pre-equilibrated with 10 column volumes of binding

buffer (50 mM NaH 2 PO 4 , pH 8.0, 500 mM NaCl, 10 mM

imidazole, 20 mM -mercaptoethanol and 10% glycerol).

The column was washed with the binding buffer at a flow

rate of 1 ml min −1 until the A 280 reached the baseline, followed

by 30 column volumes of washing buffer (50 mM

NaH 2 PO 4 , pH 6.0, 1 M NaCl, 20 mM imidazole, 20 mM -

mercaptoethanol and 10% glycerol) and 15 column volumes

of elution buffer (50 mM NaH 2 PO 4 , pH 8.0, 500 mM NaCl,

100 mM imidazole, 20 mM -mercaptoethanol, 10% glycerol)

at the same flow rate. Active fractions were pooled and

dialyzed against 1 l of gel filtration buffer (20 mM Tris–HCl,

pH 7.5 buffer containing 1 mM EDTA, 1 mM DTT, 200 mM

NaCl and 10% glycerol) for 14–16 h. The concentrated

sample (∼1 ml) was loaded onto a Sephadex G-200 column

(2.6cm × 74 cm) at a flow rate of 0.3 ml min −1 with gel

filtration buffer and fractions (4 ml) collected. Proteins were

determined by measurement of absorption at 280 nm and by

SDS–PAGE analysis. Fractions with pfPPPK-DHPS were

pooled and dialyzed against 20 mM Tris–HCl buffer, pH 8.0

containing 10% glycerol. The samples were concentrated

and small aliquots were dispensed in microcentrifuge tubes

and stored at −80 ◦ C until use. All the enzyme preparation

and purification processes were carried out at 4 ◦ C.

2.5. Enzyme assay

The activities of pfPPPK-DHPS were determined radioactively

according to the published procedure [12,15].

The assay was based on direct determination of the

amount of [ 14 C] dihydropteroate formed by the reaction

catalyzed by the enzyme DHPS, which could be separated

from [ 14 C] pABA by paper chromatography. First,

6-hydroxymethylpterin (PtCH 2 OH) was reduced with

sodium dithionite to 6-hydroxymethyl-7,8-dihydropterin

(H 2 PtCH 2 OH), the substrate of PPPK enzyme. The purity

of H 2 PtCH 2 OH was determined enzymatically using ε 254

in 0.1N HCl = 18,600 cm −1 [5]. The standard reaction

(50 l) was composed of 100 mM Tris–HCl, pH 9.0, 10 mM

MgCl 2 , 100 mM -mercaptoethanol, 10 mM ATP, 20 M

H 2 PtCH 2 OH, 100 gml −1 BSA, 10 M [ 14 C] pABA

(58 mCi mmol −1 ) and enzyme. To determine the pH profile

of the enzyme, stock solution (10×) of Tris buffer having

pH ranged from 8.0 to 10.5 were prepared and used in the

reaction. The reaction was initiated by addition of enzyme,

and incubated at 37 ◦ C for 15 min followed by placing the

tube in boiling water for 2 min to stop the reaction. Control

reactions contained all of the reagents except for the enzyme.

Precipitated proteins were removed by centrifugation

at 10,000 × g for 30 min. The clear supernatant (40 l) was

spotted on a sheet of Whatman No. 3 MM chromatography

paper (2 cm ×2cm×17.5 cm), and ascending chromatography

was performed using 0.1 M potassium phosphate buffer,

pH 7.0 at room temperature. The origin of the spot of each

chromatogram (2 cm × 2 cm) was excised, moistened with

7 ml of counting cocktail composed of 0.03% (w/v) POPOP,

0.5% (w/v) PPO in toluene solution. The radioactivity was

counted using Beckman LS3801 scintillation counter.

2.6. Kinetics and inhibition studies

Steady-state kinetic parameters of pfPPPK-DHPS for

the substrates were determined by assaying the activity of

pfPPPK-DHPS in the presence of varying concentrations

of H 2 PtCH 2 OH (0.3–50 M), [ 14 C] pABA (0.5–40 M),

ATP (2–500 M) and MgCl 2 (0.2–5 mM) while maintaining

the concentration of the other substrates. The amount of

enzyme used in each reaction was 0.007–0.008 U. Kinetic

parameters were analyzed by KALEIDAGRAPH Version

3.0.2 (Macintosh) using a non-linear least square fit of the

data to the Michaelis–Menten equation. Inhibition constants

(K i ) of the inhibitors were determined in the presence of

different inhibitor concentrations under standard assay conditions,

assuming the inhibitors were competitive. The IC 50

values (the concentration of inhibitor which inhibited 50%

of the enzyme activity) were calculated from the equation:

IC 50 = K i (1 + ([S]/K m )) in which K i is the inhibition constant

(M), [S] is the concentration of [ 14 C] pABA (M)

and K m is the Michaelis–Menten constant for pABA (M).

All the inhibitors used in the present study were dissolved

in dimethyl sulfoxide (DMSO), of which the final concentration

in the assay reaction was kept constant at 2%. Each

data point was performed in duplicate and the mean and

S.D. values were calculated. One unit of pfPPPK-DHPS

activity was defined as the amount of enzyme required to

produce 1 nmol of dihydropteroate per min at 37 ◦ C.

2.7. Cell-based inhibition studies

Methods to assess the inhibitory effects of sulfa drugs on

pfPPPK-DHPS function were developed in this study. The

cell suspension from a fresh overnight culture was first diluted

with LB media to A 600 ∼ 0.5. Twenty microliters of

the cell suspension was added to 2 ml of minimal media

(MM; 1 l of MM contained 7 g of K 2 HPO 4 ,2gofKH 2 PO 4 ,


46 W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53

0.5 g of sodium citrate, 0.1 g of Mg 2 SO 4 ,1gof(NH 4 )SO 4 ,

and 0.0025 mg ml −1 each of proline, arginine, methionine,

leucine, histidine and threonine) supplementing with varying

concentrations (0.03 M to 2 mM) of sulfa drugs dissolved

in DMSO. The final concentration of DMSO was

kept constant at 0.5% in all the reactions. The culture was

grown at 37 ◦ C with agitation at 250 rpm for 6 h prior to

measuring the A 600 . The percent survival (calculated from

the A 600 values) was plotted against log of drug concentration.

The concentrations of sulfa drug which inhibited 50%

of cell growth (IC 50 ) were calculated using GraphPad Prism

software version 2.01 and the non-linear regression curve fit

and sigmoidal curve response for drug inhibition were determined.

The percentage of cell survival in the presence of

sulfa inhibitors was compared with the control cells in the

absence of inhibitors.

3. Results

3.1. Complementation and expression of pfPPPK-DHPS

In order to facilitate purification, the carboxyl terminus

of the pKOS-pfPPPK-DHPS was fused to histidine tag to

yield pKOS-pfPPPK-DHPS(His). Transformation of DHPSdeficient

E. coli C600folP::Km r [11] with either pKOSpfPPPK-DHPS

or pKOS-pfPPPK-DHPS(His) showed that

the transformed bacteria could grow on minimal agar without

supplements, suggesting the expression of catalytically

active pfDHPS (Fig. 1). However, SDS–PAGE analysis of

the level of pfDHPS in the crude extract (∼0.2Umg −1 )

suggested that the protein was expressed at low levels.

The growth of E. coli C600folP::Km r harboring pKOSpfPPPK-DHPS

was comparable to that harboring pKOSpfPPPK-DHPS(His)

(Fig. 1B). There was no significant

difference between the pfDHPS activity in the crude extract

of E. coli C600folP::Km r harboring the two constructs

(data not shown).

3.2. Protein expression and purification

Initial attempts to express pfPPPK-DHPS at 37 ◦ C failed

to detect DHPS activity in the crude extract of the transformed

bacteria. Induction at 30 ◦ C for 12 h yielded the

highest DHPS activity in the crude extract. Lowering the

temperature to 20 ◦ C or prolonging the induction time to

>12 h did not improve the level of enzyme expression, but

rather decreased enzyme activity. Under the optimized condition,

the expressed pfPPPK-DHPS could be visualized on

a Coomassie-stained SDS–PAGE as a thin protein band with

apparent molecular mass of ∼83 kDa (Fig. 2, lane 3). This is

in agreement with the mass of 84.2 kDa calculated from the

amino acid composition of the enzyme with the histidine tag

(2136 bp; 712 aa). Based on the DHPS activity in the crude

extract and the expressed protein product on SDS–PAGE, it

is estimated that pfPPPK-DHPS was expressed at a level of

1–2% of the total soluble protein in the crude extract.

The pfPPPK-DHPS enzyme was purified using a two-step

purification scheme employing Ni-NTA affinity chromatography

followed by Sephadex G-200 chromatography. The

Fig. 1. Genetic complementation for P. falciparum PPPK-DHPS function. E. coli C600folP::Km r harboring (1) pKOS007-90PL, (2) pKOS-pfPPPK-DHPS,

(3) pKOS-pfPPPK-DHPS(His) were streaked on (A) LB agar plate and (B) minimal agar plate containing 100 gml −1 ampicillin and 40 gml −1

kanamycin. The plates were incubated at 37 ◦ C for 14–16 h before taking the photographs.


W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53 47

Fig. 2. Expression of P. falciparum PPPK-DHPS. Coomassie-stained SDS–PAGE (12%) of protein samples from IPTG-induced E. coli C600folP::Km r

harboring pKOS-pfPPPK-DHPS(His). Lane 1, molecular size markers; lane 2, pellet fraction from crude extract; lane 3, supernatant fraction from crude

extract; lane 4, proteins after Ni-NTA affinity chromatography; lane 5, proteins after Sephadex G-200 chromatography. Approximately 10 g of protein

was loaded in each lane.

majority of impurities were removed at the step of the Ni-

NTA column (Fig. 2, lanes 3 and 4), with pfPPPK-DHPS

migrating as an intense protein band of ∼83 kDa molecular

mass (Fig. 2, lane 4). Additional protein impurities were

removed by Sephadex G-200, a step which improved the

purity of pfPPPK-DHPS to ∼90% (Fig. 2, lane 5). Table 1

summarizes the fold purification and the yield of protein obtained.

The overall yield of pfPPPK-DHPS after a two-step

purification was about 2–3 mg l −1 of E. coli culture and this

amount was sufficient for characterization and scale up to

attempt crystallization studies.

3.3. Characterization of pfPPPK-DHPS

Amino-terminal sequence analysis of the electroblotted

protein band (∼83 kDa) revealed that the first 10 amino

acids, i.e. M-E-T-I-Q-E-L-I-L-S, were a perfect match for

the pfPPPK-DHPS sequence previously reported [14], confirming

that the protein was recombinant pfPPPK-DHPS.

Gel filtration using Superose 6 Prep Grade FPLC column

revealed an apparent molecular mass of ∼330 kDa (Fig. 3A

and B), suggesting that the native enzyme might be a

tetramer of identical subunit. The recombinant pfPPPK-

DHPS had a calculated pI 7.11 based on the deduced

amino acid sequence. The enzyme was activated at basic

pH (pH 9.0 to 10.5), and reached its maximal activity at

pH 10.0 (Fig. 4A). The optimal temperature of the enzyme

ranged from 37 to 45 ◦ C, beyond which the activity declined

abruptly and was completely inactivated above 55 ◦ C

(Fig. 4B). Low salt concentrations, i.e. NaCl at 0.2 M and

KCl at 0.4 M, activated the activity of pfPPPK-DHPS while

high salt concentrations inhibited the enzyme (Fig. 4C).

The activity of pfPPPK-DHPS was inactivated by low concentration

of guanidine–HCl and urea; the concentrations

Table 1

Summary of purification P. falciparum PPPK-DHPS a

Step Protein (mg) Total activity (nmol min −1 ) Specific activity (nmol min −1 mg −1 ) Fold Percent yield

Crude extract 512 110 0.21 1 100

Ni-NTA 9.1 79 8.7 41 72

Sephadex G-200 3.4 36 10.6 50 33

a From 1 l of E. coli culture.


48 W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53

Fig. 3. Native molecular mass of P. falciparum PPPK-DHPS. FPLC profile of the recombinant pfPPPK-DHPS on Superose 6 prep grade prepacked column

HR 16/50 (Pharmacia). The column was run at flow rate 0.3 ml min −1 using 20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 200 mM NaCl, 10%

glycerol as the buffer. Fractions of 1.5 ml were collected. (A) Absorbance at 280 nm () and activity of pfPPPK-DHPS () determined radioactively

according to the procedure described in the text. (B) Partition coefficient (K av ) plotted against log molecular mass of standard proteins. Arrow indicates

the native pfPPPK-DHPS (∼330 kDa).

of guanidine–HCl and urea required to inhibit 50% of the

pfPPPK-DHPS were 0.25 and 0.9 M, respectively (Fig. 4D).

The activity of the recombinant pfPPPK-DHPS declined

rapidly when the storage temperature was increased (data

not shown). No loss of pfPPPK-DHPS activity was observed

when the enzyme was stored at −20 and −80 ◦ C for 3

months in 20 mM Tris–HCl, pH 8.0 buffer containing 10 or

20% glycerol. In buffer without glycerol, 30 and 50% of the

enzyme activity were lost when stored for 6 months at −80

and −20 ◦ C, respectively (Table 2).

3.4. Enzyme kinetics and inhibition studies

Steady-state kinetic parameters of pfPPPK-DHPS were

determined for H 2 PtCH 2 OH, pABA, ATP, and Mg 2+ . The

apparent K m values for H 2 PtCH 2 OH, pABA, ATP and Mg 2+

were 1.22 ± 0.11 M, 1.25 ± 0.10 M, 11.9 ± 1.1 M and

0.92±0.10 mM, respectively. Double reciprocal plots clearly

revealed that sulfathiazole and sulfadoxine, competitively

Table 2

Kinetic properties of P. falciparum PPPK-DHPS

Kinetic parameters

K m

H 2 PtCH 2 OH (M) 1.22 ± 0.11

pABA (M) 1.25 ± 0.10

ATP (M) 11.9 ± 1.12

Mg 2+ (mM) 0.92 ± 0.10

Optimal temperature ( ◦ C) 37–45

Optimal pH 10.0

Activator Mg 2+

k cat (s −1 ) 0.03

k cat /K m (M −1 s −1 ) 2.4 × 10 4

Effect of NaCl and KCl Activation at low concentrations

Inhibition at high concentrations

Inhibition by urea and

Yes

guanidine–HCl

Thermal stability

Sensitive to high temperature

Storage stability a

No loss of activity for 3-month

storage in 20 mM Tris–HCl, pH

8.0 containing 10 or 20% glycerol

a Storage conditions were examined at −20 and −80 ◦ C.


W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53 49

Fig. 4. Effects of pH, temperature, salts, urea and guanidine HCl. The activities of pfPPPK-DHPS were determined at pH 8.0–10.5 (A) and temperature

25–55 ◦ C (B). The effects of KCl and NaCl (C) and urea and guanidine HCl (D) were investigated. The percentage of activities was calculated in relation

to the maximal activities (100%). The data were average values from two independent experiments.

inhibited the pfPPPK-DHPS (Fig. 5A and B). Sixteen sulfa

derivatives were investigated with respect to their abilities

to inhibit the enzyme pfPPPK-DHPS (Table 3). Sulfadoxine,

an inhibitor used in a fixed-dose combination with antifolate

pyrimethamine in the treatment of uncomplicated P.

falciparum infection, inhibited pfPPPK-DHPS with the K i

value of 11.0 ± 0.4 M. Except for sulfanilamide and sulfacetamide

whose binding affinities were ∼4 and 9-fold, respectively,

poorer than that of sulfadoxine, the other sulfa

derivatives were found to bind pfPPPK-DHPS with ∼2- to

20-fold greater affinity than that of sulfadoxine (Table 3).

3.5. Cell-based inhibition by sulfa drugs

The ability of the sulfa derivatives to inhibit growth of

bacterial cells harboring pKOS-pfPPPK-DHPS(His) was investigated

in liquid media. Out of 16 derivatives examined,

12 were found to be potent inhibitors of bacterial growth

with IC 50 values ranging from 1.5 to 28.5 M (Table 3).

Sulfadoxine, sulfanilamide and sulfacetamide were moderately

potent inhibitors with the IC 50 values of 100–160 M

(Table 3). The poorest inhibitor among these 16 sulfa

derivatives in the cell-based assay was p-aminosalicylic acid

(PAS). A correlation between cell-based and enzyme-based

inhibition is shown in Fig. 6.

4. Discussion

Initial attempts to express the bifunctional pfPPPK-DHPS

under the control of T5 promoter at 37 ◦ C yielded a faint

band of ∼83 kDa on Coomassie-stained SDS–PAGE. Unfortunately,

these conditions yielded expressed product that

was insoluble and associated with inclusion bodies. The optimal

condition that gave soluble enzyme with the highest

DHPS activity was 30 ◦ C for 12 h. The level of expression of

pfPPPK-DHPS was low but this seems to be common problem

for most of the malarial genes that are difficult to express.

Low expression of malarial genes could be due to: (1)

the high genetic AT content (∼80%) which causes heterologous

expression of P. falciparum proteins extremely difficult

in commonly used prokaryotic and eukaryotic expression

systems [24]; (2) the production of unstable or toxic protein

[25]; (3) the inefficient transcription and/or translation


50 W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53

Fig. 5. Lineweaver–Burk plots of pfPPPK-DHPS inhibited by sulfa inhibitors. The assays were performed as described in the text in the presence

of varying concentrations of pABA while keeping the concentration of H 2 PtCH 2 OH constant at 20 M: (A) sulfathiazole and (B) sulfadoxine. The

concentrations of the inhibitors (M) are as specified.

and degradation of mRNA and/or expressed protein in

bacteria.

Purification of pfPPPK-DHPS was accomplished by two

sequential chromatographic steps; Ni-NTA affinity chromatography

followed by Sephadex G-200 gel filtration.

Generally the yield of enzyme was ∼3mgl −1 of E. coli

culture. This is a great improvement over the yields reported

in the previous study [15] in which only 0.05–0.1 mg l −1

was obtained (unpublished observation). The presence of

glycerol at a final concentration of 10% or more during the

purification steps is critical for the stability of the enzyme.

The Ni-NTA step removed most of the impurities (Fig. 2,

lane 4). However, there remained low molecular weight proteins

that were co-eluted with the pfPPPK-DHPS enzyme.

These proteins were subsequently identified to be E. coli

origin and could generally be removed by gel filtration, except

for the proteins of molecular mass 36 and 48 kDa that

always co-migrated with pfPPPK-DHPS. Surprisingly, the

observation that the first 10 residues from the N-terminus

of the 36 kDa protein perfectly match with the first 10 N-

terminal amino acids of the recombinant pfPPPK-DHPS

suggested that the 36 kDa protein possessed intact native

N-terminus but for some unclear mechanism was truncated

at its C-terminus. Whether the truncation is associated with

erroneous translational termination or cleavage of protein

at the site sensitive to proteases [26] remains to be further

investigated. The reactivity of the expressed protein

with monoclonal antibody against penta-histidines (data not

shown) may be attributed to histidine residues within the

interdomain region of the enzyme. Our system is currently

the best resource that provides an abundant supply of active

pfPPPK-DHPS for crystallization trials and drug screening.

The gel filtration data of the recombinant pfPPPK-DHPS

revealed that the molecular mass of the enzyme (∼330 kDa)


W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53 51

Table 3

Inhibition of P. falciparum PPPK-DHPS by sulfa inhibitors

Sulfa derivatives Structure Enzyme inhibition Cell-based inhibition IC 50 (M) b

K i (M) a

IC 50 (M)

Sulfathiazole 0.6 ± 0.02 5.2 1.5

Dapsone 1.0 ± 0.1 8.8 4.9

Sulfachloropyridazine 1.5 ± 0.03 12.6 3.0

Sulfaquinoxaline 1.6 ± 0.04 13.8 28.5

Sulfadimethoxine 1.8 ± 0.03 15.9 16.3

Sulfapyridine 1.9 ± 0.10 16.6 4.7

Sulfamoxole 2.7 ± 0.05 23.2 8.0

Sulfamethoxypyridazine 3.3 ± 0.15 28.6 4.7

Sulfamethoxazole 4.4 ± 0.13 38.4 3.5

Sulfamerazine 4.8 ± 0.3 41.9 4.7

Sulfadiazine 6.8 ± 0.24 58.7 8.3

Sulfisoxazole 7.6 ± 0.4 66.3 15.0

PAS 8.2 ± 0.5 71.6 18.270

Sulfadoxine 11.0 ± 0.4 95.1 150.3

Sulfanilamide 43.7 ± 3.0 380.0 160.3

Sulfacetamide 94.1 ± 4.5 818.0 100.2

a Calculated from non-linear least square analysis for competitive inhibition.

b Estimated values from plots between percent survival vs. log[drug concentration].

was slightly larger than that previously reported [15]. The

discrepancies could be due to the diverse nature of the enzyme

in different organisms and/or perhaps attributed by the

different conditions and techniques employed in the study.

The molecular mass reported for the bifunctional PPPK-

DHPSs of other plasmodia and protozoa also varied considerably;

200–250 kDa for Plasmodium berghei [27], 190 kDa

for Plasmodium chabaudi [28] and 125 kDa for T. gondii

[16].

The presence of pfPPPK in the form of intact bifunctional

pfPPPK-DHPS is crucial for the activity of pfDHPS.

This observation could lead to further exploration of the

role of the pfPPPK and its interactions with the pfDHPS

domain which might affect the conformation of the protein.

It is also conceivable that the close proximity between

the two catalytic sites could help facilitate the transfer of

substrates and/or products of the sequential reaction in the

pathway, a hypothesis that remains to be further verified and


52 W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53

Fig. 6. Correlation between cell-based and enzyme-based inhibition by

sulfa inhibitors. Log IC 50 values determined from inhibition of the growth

of E. coli C600folP::Km r harboring pKOS-pfPPPK-DHPS(His) by sulfa

inhibitors were plotted against log IC 50 values calculated from inhibition

of the enzyme by the corresponding sulfa inhibitors. Data were analyzed

by a linear regression fitting program.

demonstrated. The K m value for pABA (1.25 ± 0.10 M) in

the present study was found to be almost two orders of magnitude

higher than the value reported in the previous study

[15]. We currently have no explanation for this difference,

but it may be attributed to the differences in the enzyme and

the purity of the substrate. It is possible that the residual of

the truncated 36 kDa protein present in the purified pfPPPK-

DHPS sample could attribute to the high apparent K m values

of the substrates in the present study. Other possible factors

could involve the differences in the assay method and assay

conditions. It is noteworthy that the K m values for pABA of

DHPSs reported for bacteria and other protozoa varied from

micromolar to submicromolar [29–32]. Notwithstanding

the different K m values for pABA, our results agree within

a factor of 2 for the specific activity with the values previously

reported for pfPPPK-DHPS. The reaction catalyzed

by pfPPPK-DHPS was both Mg 2+ - and ATP-dependent.

The involvement of ATP in the reaction implied that the

enzyme requires a phosphorylated 6-hydroxymethyl-7,8-

dihydropterin as an intermediate substrate for the reaction,

supporting the compulsory order ternary complex mechanism

proposed earlier for E. coli DHPS; PPPK binds first

to ATP in a relatively slow step, followed by the faster

addition of 6-hydroxymethyl-7,8-dihydropteroate [33].

Inhibition of pfPPPK-DHPS by sulfathiazole and sulfadoxine

confirmed that the two sulfa drugs competed for binding

with the substrate at the active site of DHPS (Fig. 5).

However, the calculated K i value of sulfadoxine was nearly

two orders of magnitude higher than that reported previously

[15]. One possible explanation of the discrepancy could be

the presence of C-terminal His-tag in the construct used

in the present study which was not present in the previous

study. The different assay conditions and the different purity

of the substrate used in the assay could be the other

possible factors attributed to the difference. Inhibition of

pfPPPK-DHPS by a wide range of sulfonamides including

a sulfone revealed that malarial enzyme has a broad range

of inhibitory potencies for these drugs (Table 3). The observations

that sulfathiazole and dapsone are potent inhibitors

of pfDHPS are in good agreement with the previous in vivo

[34] and in vitro [15] studies. Sulfadoxine, a common therapeutic

drug used for malaria prophylaxis, inhibited the bacterial

cell growth with similar effectiveness. The results from

the cell-based inhibition assays seem to correlate with those

from enzyme-based studies (Table 2 and Fig. 6). Although

the data do not take into consideration other important in

vivo factors such as membrane transport, drug stability, elimination

half-life, pharmacokinetics, pharmacodynamics, and

toxicity in human, the cell-based inhibition approach may

provide a primary guidance to selection of potential candidates

for further studies.

Acknowledgements

This work was supported in part by grants from Tropical

Disease Research (to W.S.) and The Wellcome Trust (to

W.S./A.F.C.). We thank the Royal Golden Jubilee Ph.D. programme,

Thailand Research Fund for the generous support

and the Ph.D. fellowship for Waraporn Kasekarn.

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