Molecular & Biochemical Parasitology 137 (2004) 43–53
Molecular characterization of bifunctional hydroxymethyldihydropterin
pyrophosphokinase-dihydropteroate synthase from
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
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
The parasite Plasmodium falciparum is responsible for
hundreds of millions of cases and kills approximately 2.7
millions people each year . 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
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
∗ Corresponding author. Tel.: +66 2 201 5605; fax: +66 2 248 0375.
E-mail address: email@example.com (W. Sirawaraporn).
targets for malarial chemotherapy. Hydroxymethyldihydropterin
(pfPPPK-DHPS) of P. falciparum is a target of sulfadoxine
. Formulated as a fixed-dose combination with antifolate
inhibitors such as pyrimethamine , the combination
(Fansidar ® ) has been widely exploited for the treatment of
uncomplicated “falciparum” malaria in Africa where these
parasites are resistant to chloroquine .
Hydroxymethyldihydropterin pyrophosphokinase (PPPK;
EC 188.8.131.52) 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 184.108.40.206) in the biosynthesis of
tetrahydrofolate . 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.
44 W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53
natural substrate, pABA, for binding to the enzyme ,
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 , Escherichia
coli , Neisseria meningitidis , Mycobacterium
tuberculosis , and Mycobacterium leprae .
Unlike DHPS of prokaryotes, the enzyme of protozoan parasites
such as P. falciparum [13–15] and Toxoplasma gondii
 is a bifunctional enzyme with PPPK. In Pneumocystis
carinii, however, the DHPS is a trifunctional enzyme with
dihydroneopterin aldolase (DHNA) and PPPK . 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 , 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
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
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
 was used as a template for subcloning into pKOS007-
90-PL expression vector . 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  was used as a host strain for the expression
2.3. Construction of expression plasmids
The gene encoding the wild-type pfPPPK-DHPS in pTrc
vector  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 ′ -
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
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 . 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.
3.1. 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  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 , 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
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
Kinetic properties of P. falciparum PPPK-DHPS
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
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.
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 ; (2) the production of unstable or toxic protein
; (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
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  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  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
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 . 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 , 190 kDa
for Plasmodium chabaudi  and 125 kDa for T. gondii
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
. 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 .
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
. 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
 and in vitro  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.
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.
 Breman JG. The ears of the hippopotamus: manifestations, determinants,
and estimates of the malaria burden. Am J Trop Med Hyg
 Dollery C. Therapeutic drugs: sulfadoxine. 2nd ed. Churchill Livingstone;
 Dollery C. Therapeutic drugs: pyrimethamine. 2nd ed. Churchill
 Sibley CH, Hyde JE, Sims PF, et al. Pyrimethamine-sulfadoxine
resistance in Plasmodium falciparum: what next? Trends Parasitol
 Shiota T, Disraely MN, McCann MP. The enzymatic synthesis of
folate-like compounds from hydroxyldihydropteridine pyrophosphate.
J Biol Chem 1964;239:2259–66.
 Brown GM. The biosynthesis of folic acid: II. Inhibition by sulfonamides.
J Biol Chem 1962;237:536–40.
 Lopez P, Espinosa M, Greenberg B, Lacks SA. Sulfonamide resistance
in Streptococcus pneumoniae: DNA sequence of the gene
encoding dihydropteroate syntheses and characterization of the enzyme.
J Bacteriol 1987;169:4320–6.
 Lacks SA, Greenberg B, Lopez P. A cluster of four genes encoding
enzymes for five steps in the folate biosynthetic pathway of Streptococcus
pneumoniae. J Bacteriol 1995;177:66–74.
 Slock J, Stahly DP, Han CY, Six EW, Crawford IP. An apparent
Bacillus subtilis folic acid biosynthetic operon containing pab, an
amphibolic trpG gene, a third gene required for synthesis of paraaminobenzoic
acid, and the dihydropteroate syntheses gene. J Bacteriol
 Talarico TL, Ray PH, Dev IK, Merrill BM, Dallas WS.
Cloning, sequence analysis, and overexpression of Escherichia
coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase.
J Bacteriol 1992;174:5971–7.
W. Kasekarn et al. / Molecular & Biochemical Parasitology 137 (2004) 43–53 53
 Fermer C, Swedberg G. Adaptation to sulfonamide resistance in
Neisseria meningitidis may have required compensatory changes to
retain enzyme function: kinetic analysis of dihydropteroate synthases
from N. meningitidis expressed in a knockout mutant of Escherichia
coli. J Bacteriol 1997;179:831–7.
 Nopponpunth V, Sirawaraporn W, Greene PJ, Santi DV. Cloning
and expression of Mycobacterium tuberculosis and Mycobacterium
leprae dihydropteroate synthase in Escherichia coli. J Bacteriol
 Brooks DR, Wang P, Read M, Watkins WM, Sims PF, Hyde JE.
Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase:
dihydropteroate synthase gene in lines of the human malaria
parasite, Plasmodium falciparum, with differing resistance to sulfadoxine.
Eur J Biochem 1994;224:397–405.
 Triglia T, Cowman AF. Primary structure and expression of the
dihydropteroate synthetase gene of Plasmodium falciparum. Proc
Natl Acad Sci USA 1994;91:7149–53.
 Triglia T, Menting JG, Wilson C, Cowman AF. Mutations in dihydropteroate
synthase are responsible for sulfone and sulfonamide
resistance in Plasmodium falciparum. Proc Natl Acad Sci USA
 Allegra CJ, Boarman D, Kovacs JA, et al. Interaction of sulfonamide
and sulfone compounds with Toxoplasma gondii dihydropteroate
synthase. J Clin Invest 1990;85:371–9.
 Volpe F, Ballantine SP, Delves CJ. The multifunctional folic acid
synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin
aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate
synthase. Eur J Biochem 1993;216:449–58.
 Achari A, Somers DO, Champness JN, Bryant PK, Rosemond J,
Stammers DK. Crystal structure of the anti-bacterial sulfonamide
drug target dihydropteroate synthase. Nat Struct Biol 1997;4:490–7.
 Hampele IC, D’Arcy A, Dale GE, et al. Structure and function of the
dihydropteroate synthase from Staphylococcus aureus. J Mol Biol
 Baca AM, Sirawaraporn R, Turley S, Sirawaraporn W, Hol WG.
Crystal structure of Mycobacterium tuberculosis 7,8-dihydropteroate
synthase in complex with pterin monophosphate: new insight
into the enzymatic mechanism and sulfa-drug action. J Mol Biol
 Wang P, Read M, Sims PF, Hyde JE. Sulfadoxine resistance in
the human malaria parasite Plasmodium falciparum is determined
by mutations in dihydropteroate synthetase and an additional factor
associated with folate utilization. Mol Microbiol 1997;23:979–
 Triglia T, Cowman AF. The mechanism of resistance to sulfa drugs
in Plasmodium falciparum. Drug Resist Updates 1999;2:15–9.
 Le Bras J, Durand R. The mechanisms of resistance to antimalarial
drugs in Plasmodium falciparum. Fundam Clin Pharmacol
 Withers-Martinez C, Carpenter EP, Hackett F, et al. PCR-based gene
synthesis as an efficient approach for expression of the A + T-rich
malaria genome. Protein Eng 1999;12:1113–20.
 Hall SJ, Sims PF, Hyde JE. Functional expression of the dihydrofolate
reductase and thymidylate synthetase activities of the human malaria
parasite Plasmodium falciparum in Escherichia coli. Mol Biochem
 Schmincke-Ott E, Bisswanger H. Multifunctional proteins: introduction.
New York: Wiley; 1980.
 Ferone R. The enzymic synthesis of dihydropteroate and dihydrofolate
by Plasmodium berghei. J Protozool 1973;20:459–64.
 Walter RD, Konigk E. 7,8-Dihydropteroate-synthesizing enzyme
from Plasmodium chabaudi. Methods Enzymol 1980;66:564–70.
 Ortiz PJ. Dihydrofolate and dihydropteroate synthesis by partially
purified enzymes from wild-type and sulfonamide-resistant pneumonococcus.
 McCullough JL, Maren TH. Inhibition of dihydropteroate synthetase
from Escherichia coli by sulfones and sulfonamides. Antimicrob
Agents Chemother 1973;3:665–9.
 McCullough JL, Maren TH. Dihydropteroate synthetase from Plasmodium
berghei: isolation, properties, and inhibition by dapsone and
sulfadiazine. Mol Pharmacol 1974;10:140–5.
 Walter RD, Konigk E. Biosynthesis of folic acid compounds in
plasmodia. Purification and properties of the 7,8-dihydropteroatesynthesizing
enzyme from Plasmodium chabaudi. Hoppe Seylers Z
Physiol Chem 1974;355:431–7.
 Bermingham A, Bottomley JR, Primrose WU, Derrick JP. Equilibrium
and kinetic studies of substrate binding to 6-hydroxymethyl-
7,8-dihydropterin pyrophosphokinase from Escherichia coli. J Biol
 Zhang Y, Meshnick SR. Inhibition of Plasmodium falciparum dihydropteroate
synthetase and growth in vitro by sulfa drugs. Antimicrob
Agents Chemother 1991;35:267–71.