Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: A unique bifunctional enzyme from Plasmodium falciparum

Article abstract of DOI:10.1042/BJ20110170

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper,we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs. The Authors Journal compilation

Full text of DOI:10.1042/BJ20110170

Biochem. J. (2011) 436, 641–650 (Printed in Great Britain) doi:10.1042/BJ20110170
641
Glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase:
a unique bifunctional enzyme from Plasmodium falciparum
Esther JORTZIK*1, Boniface M. MAILU*1, Janina PREUSS*†, Marina FISCHER*, Lars BODE†, Stefan RAHLFS* and
Katja BECKER*2
*Interdisciplinary Research Center, Justus Liebig University, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany, and †Department of Pediatrics, University of California San Diego,
200 West Arbor Drive, San Diego, CA 92103-8450, U.S.A.
The survival of malaria parasites in human RBCs (red blood cells)
depends on the pentose phosphate pathway, both in Plasmodium
falciparum and its human host. G6PD (glucose-6-phosphate
dehydrogenase) deficiency, the most common human enzyme
deficiency, leads to a lack of NADPH in erythrocytes, and
protects from malaria. In P. falciparum, G6PD is combined
with the second enzyme of the pentose phosphate pathway to
create a unique bifunctional enzyme named GluPho (glucose-
6-phosphate dehydrogenase–6-phosphogluconolactonase). In the
present paper, we report for the first time the cloning, heterologous
overexpression, purification and kinetic characterization of both
enzymatic activities of full-length PfGluPho (P. falciparum
GluPho), and demonstrate striking structural and functional
differences with the human enzymes. Detailed kinetic analyses
indicate that PfGluPho functions on the basis of a rapid
equilibrium random Bi Bi mechanism, where the binding of the
second substrate depends on the first substrate. We furthermore
show that PfGluPho is inhibited by S-glutathionylation. The
availability of recombinant PfGluPho and the major differences
to hG6PD (human G6PD) facilitate studies on PfGluPho as an
excellent drug target candidate in the search for new antimalarial
drugs.
Key words: glucose 6-phosphate dehydrogenase, pentose
phosphate pathway, 6-phosphogluconolactonase, Plasmodium,
malaria, redox.
[5]; (ii) malaria parasites require NADPH produced in the PPP;
and (iii) the PPP in Plasmodium is highly regulated in different
developmental stages and by the level of oxidative stress [6].
The existence of a G6PD in malaria parasites had been contro-
versially discussed, until the enzyme could be partially purified
from infected RBCs [7,8]. In contrast with the PPP enzymes in
vertebrates and bacteria, the first two enzymes of the Plasmodium
PPP are combined in a unique bifunctional enzyme of 910
amino acids called GluPho (glucose-6-phosphate dehydrogenase–
6-phosphogluconolactonase) [9,10] (Supplementary Figure S1
at http://www.BiochemJ.org/bj/436/bj4360641add.htm). The C-
terminal part (amino acids 311–911) of GluPho is homologous
with other G6PDs, although interrupted by an insertion of 62
amino acids [9], whereas the N-terminal 310 amino acids show
a high similarity to 6PGL [11]. The insertion in the G6PD part
is highly conserved among Plasmodium, and is essential for the
G6PD activity of Plasmodium berghei GluPho [12].
The importance of PfGluPho (Plasmodium falciparum GluPho)
is further substantiated by the finding that RNA-mediated gene
silencing results in arrest at the trophozoite stage and enhanced
gametocyte formation [13]. However, this result has to be handled
with care, since the existence of RNAi (RNA interference)
in Plasmodium is questionable [14,15]. Nonetheless immediate
transcript enhancement of thioredoxin reductase accompanies
PfGluPho knockdown and suggests a central role of PfGluPho
in the response towards oxidative stress [13].
INTRODUCTION
G6PD (glucose-6-phosphate dehydrogenase) deficiency is the
most common human enzyme defect and is associated with
resistance to malaria, most probably due to decreased levels of
reducing equivalents in the form of NADPH [1]. Malaria is one
of the most severe infectious diseases with 240 million cases in
2009 (http://www.who.int/malaria/world_malaria_report_2010/
worldmalariareport2010.pdf). The increasing resistance of
Plasmodium falciparum to antimalarial drugs, including the
artemisinin-based combination treatments, intensifies the search
for new antimalarial drug targets [2,3].
The PPP (pentose phosphate pathway) is the major source of
NADPH and pentose sugars, which are crucial for oxidative stress
defence and nucleotide synthesis. Enhanced oxidative
stress induced by G6PD deficiency or pro-oxidant antimalarial
compounds such as 4-aminoquinolines (e.g. chloroquine) and
8-aminoquinolines (e.g. primaquine), as well as artemisinins,
suppresses parasite growth and enhances the elimination of
parasitized RBCs (red blood cells) by the immune system (for a
review see [4]).
The first steps of the PPP are catalysed by G6PD (EC 1.1.1.49),
6PGL (6-phosphogluconolactonase; EC 3.1.1.31), and 6PGD (6-
phosphogluconate dehydrogenase; EC 1.1.1.44). Previous studies
have suggested that the PPP of both the human host and the
malaria parasite are important during the infection of RBCs with
Plasmodium: (i) hG6PD (human G6PD) deficiency and thus a re-
stricted PPP limits NADPH production and protects from malaria
The outmost importance of the parasite’s PPP in the parasite–
host unit suggests that PfGluPho is a most attractive drug
Abbreviations used: DTT, dithiothreitol; G6P, glucose 6-phosphate; GluPho, glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase;
G6PD, glucose-6-phosphate dehydrogenase; hG6PD, human G6PD; IPTG, isopropyl-β-D-thiogalactopyranoside; MALDI, matrix-assisted laser-desorption
ionization; NIH, National Institutes of Health; PfGluPho, Plasmodium falciparum GluPho; 6PGD, 6-phosphogluconate dehydrogenase; 6PGL,
6-phosphogluconolactonase; h6PGL, human 6PGL; 6PGγL, 6-phosphoglucono-γ-lactone; 6PGδL, 6-phosphoglucono-δ-lactone; PPP, pentose phosphate
pathway; RBC, red blood cell; TOF, time-of-flight.
1
These authors contributed equally to this work.
To whom correspondence should be addressed (email katja.becker@ernaehrung.uni-giessen.de).
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E. Jortzik and others
restriction sites (underlined) (forward: 5ꢁ-CGCGCATATGGC-
CGCGCCGGCCCCG-3ꢁ; reverse: 5ꢁ-GCGCGGATCCCTACAA-
AGTGGAATGCTTCTCGAA-3ꢁ) and cloned into the expression
vector pET28a (Novagen) with an N-terminal His tag.
target [16]. In a recent screening approach, 172 out of around
300000 chemical compounds were identified to be active
against P. falciparum in vitro. Interestingly, two compounds
with antimalarial activity (C276-1187 and D052-0147; Chemdiv)
bound to the separately cloned 6PGL part of PfGluPho (referred to
as 6PGLPfGluPho) in thermal melt-shift assays, thereby underlining
the potential of PfGluPho as an antimalarial drug target [17].
Until now, analyses of PfGluPho were restricted because the
full-length gene could not be cloned [10]. Previous functional
studies focused on the corresponding enzyme of the rodent
parasite P. berghei [10,12]. In the present paper we report for
the first time the successful production of the complete active
recombinant GluPho from P. falciparum. Biochemical analyses
reveal unique structural and functional features of PfGluPho,
which clearly distinguish the bifunctional enzyme from the mono-
functional human enzymes and provide a basis for the
development of new therapeutic agents.
Heterologous overexpression and purification
The highest yield of recombinant PfGluPho was obtained by
heterologous overexpression of PfGluPho in the vector pQE30 in
Escherichia coli M15 cells (Qiagen) with pRAREII (Novagen).
The cells were grown in Terrific Broth (12 g of tryptone, 24 g
of yeast extract, 9.4 g of K2HPO4 and 4 ml of glycerol per litre
of medium) medium supplemented with carbenicillin, kanamycin
(both 50 μg/ml) and chloramphenicol (12.5 μg/ml) at 23◦C to
an attenuance at 600 nm (D600) of 0.6, and the expression was
induced by 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside).
After 24 h, the cells were harvested by centrifugation (15 min
at 6000 g and 4◦C), resuspended in 5 ml of 0.1 M Tris/HCl
(pH 7.8) and 0.5 M NaCl per g of cell pellet, and mixed
with protease inhibitors (50 μM phenylmethylsulfonyl fluoride,
150 nM pepstatin and 40 nM cystatin). The cells were lysed by
lysozyme and DNAse for 2 h at 4◦C, sonicated (three cycles of
20 s at 70% and 4 ◦C), and centrifuged (30 min at 30600 g
and 4◦C). The supernatant was applied to a Ni-NTA column
(Qiagen) and recombinant proteins were eluted with 0.1 M
Tris/HCl (pH 7.8) and 0.5 M NaCl containing 250 mM imidazole.
The purity of protein samples was controlled by SDS/PAGE
(12% gel). The protein was further purified by gel-filtration
chromatography.
E. coli M15 cells containing the plasmids pQE30 and
pRAREII (Novagen) were grown in Terrific Broth medium
supplemented with carbenicillin, kanamycin (both 50 μg/ml) and
chloramphenicol (12.5 μg/ml) at 23◦C, and the expression was
induced at D600 of 0.6 with 0.5 mM IPTG. The cells were harvested
after 20 h, and resuspended as described for PfGluPho. The cells
were lysed for 1 h at 4◦C by lysozyme and DNase, sonicated
(three cycles of 20 s at 70% and 4 ◦C), and centrifuged (30 min
at 30600 g and 4◦C). Recombinant proteins were eluted from a
Ni-NTA column with 0.1 M Tris/HCl (pH 7.8) and 0.5 M NaCl
containing 0.3 M imidazole yielding 1.5 mg of pure protein per
litre of E. coli culture.
Overexpression of hG6PD was performed in E. coli BL21 cells
(Invitrogen) containing pRAREII in 2×YT (16 g of tryptone,
10 g of yeast and 5 g of NaCl per litre of medium) medium
with kanamycin (50 μg/ml) and chloramphenicol (12.5 μg/ml) at
23◦C. At a D600 of 1, the expression was induced by 0.1 mM IPTG
and continued for 24 h. Harvesting, cell lysis and purification in
50 mM Tris/HCl (pH 8.0), 300 mM NaCl and 0.1 mM NADP+
were performed as described for G6PDPfGluPho. hG6PD can be
eluted from the Ni-NTA column with buffer containing 150 and
200 mM imidazole.
h6PGL was overexpressed in E. coli BL21/pRAREII in Terrific
Broth medium supplemented with kanamycin (50 μg/ml) and
chloramphenicol (12.5 μg/ml) at 37◦C. The expression was
induced at a D600 of 0.6 with 1 mM IPTG, and the cells were
harvested 4 h after induction as described above. The cells
were resuspended in 100 mM triethanolamine and 350 mM NaCl
(pH 7.4). h6PGL was purified as described for G6PDPfGluPho
and eluted from the Ni-NTA column at 50–200 mM imidazole
yielding 30 mg of pure protein per litre of E. coli culture.
MATERIALS AND METHODS
PCR amplification, sequencing and cloning of PfGluPho,
G6PDPfGluPho, hG6PD and h6PGL
The gene of PfGluPho (PlasmoDB accession number PF14_0511)
was identified on chromosome 14 and amplified by PCR on
a P. falciparum 3D7 gametocyte cDNA library. Perfect
match primers (forward: 5ꢁ-CGCGGGATCCGATTATGAGA-
ATTTTGTAAAAAGTGCAG-3ꢁ; reverse: 5ꢁ-GCGCAAGCTT-
TCAATTAATATCTAACAAATCGTCTTC-3ꢁ; MWG-Biotech)
introduced BamHI and HindIII restriction sites (underlined).
Cloning of the complete PfGluPho gene was not successful;
therefore we cloned the gene in two separate parts. A silent
mutation was introduced to create a SacI restriction site
within the PfGluPho gene. The first part was amplified
using the same BamHI forward restriction site primer and a
reverse primer containing a SacI restriction site (underlined)
(5ꢁ-GCGCGAGCTCTTCTTTATTCAAACTATTAGAATAAAG-
AG-3ꢁ); the second part was amplified using
a SacI
restriction site primer (5ꢁ-GCGCGAGCTCTTAACTATAA-
TAATTTTTGGCTGTTCAG-3ꢁ) and the reverse primer
containing a HindIII restriction site. Both constructs were
separately cloned into a pSK vector (Stratagene). A triple ligation
using the vector pET28a and the two parts of PfGluPho was
performed to successfully combine the PfGluPho parts in the
vector pET28a. For optimization of heterologous overexpression,
the complete PfGluPho construct was also cloned into the vector
pQE30 (Qiagen). Both constructs contain an N-terminal His tag.
The G6PD part of PfGluPho ranging from amino acid 339
to 910 was cloned as described for PfGluPho (forward: 5ꢁ-CG-
GGATCCACTATAATAATTTTTGGCTGTTCAG-3ꢁ, reverse: 5ꢁ-
GCAAGCTTTCAATTAATATCTAACAACTCGTC-3ꢁ; BamHI
and Hind III restriction sites underlined).
A
clone of hG6PD (GenBank® accession number
NP_001035810) from the NIH (National Institutes of Health)
Mammalian Gene Collection (clone ID 282264) was purchased
from Invitrogen. The gene was amplified by PCR using primers
designed to introduce restriction sites for NdeI and XhoI (under-
lined) (forward: 5ꢁ-GCGCCATATGGCAGAGCAGGTGGCCCT-
3ꢁ; reverse: 5ꢁ-CGCGCTCGAGGAGCTTGTGGGGGTTCACC-
3ꢁ) and the construct was cloned into the expression vector pET24a
(Novagen) with a C-terminal His tag.
Protein immunoblotting analyses
A
clone of h6PGL (GenBank® accession number
NP_036220.1) from the NIH mammalian gene collection (clone
ID 4053022) was purchased from Invitrogen. The gene was
amplified by PCR using primers that introduce NdeI and BamHI
For the recombinant PfGluPho, semi-dry Western blots using
an anti-His6 antibody (Dianova) and a phosphatase-conjugated
goat anti-mouse antibody (Dianova) were performed. Specific
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643
antibodies for PfGluPho were obtained from rabbits that
had been immunized with synthetic peptides of PfGluPho:
an N-terminal peptide composed of amino acids 118–133
(KEQLYKPDTTKSIVDC; anti-Pho) and a C-terminal peptide
composed of amino acids 896–910 (CVRKSSFYEDDLLDIN;
anti-Glu) (Eurogentec, diluted 1:5000). Horseradish peroxidase-
conjugated anti-rabbit antibody (Dianova, diluted 1:50000) was
used as the secondary antibody.
concentrations of S1. Rearrangement of the equation shows that
the intercepts of primary double-reciprocal plots with 1/[S2]
as the variable are given by ꢀ0 + ꢀS1/[S1] and the slopes by
ꢀS2 + ꢀS2S1/[S1]. The secondary plots of the intercepts and slopes
against 1/[S1] provide estimates for the individual-rate parameters
[19].
Analogous kinetic experiments were carried out with hG6PD
to allow direct comparison with the Plasmodium enzyme.
Alternative substrate and inhibition studies
Gel-filtration chromatography
The steady-state kinetics of PfGluPho’s G6PD activity, as well as
for hG6PD using the G6P analogue 2deoxyG6P (Sigma–Aldrich),
were performed by varying the concentration of 2deoxyG6P from
1–10 mM in the presence of NADP+ at the Km, whereas NADP+
was varied from 1–200 μM with the concentration of 2deoxyG6P
at Km.
In product inhibition studies with PfGluPho and hG6PD, the
initial rates were measured for a series of NADPH concentrations
(0–30 μM) with 60 μM G6P and NADP+ concentrations from 2–
200 μM. Likewise the experiment was carried out by varying the
G6P concentrations from 5–200 μM and NADPH (0–20 μM),
whereas fixing the concentration of NADP+ at 10 μM. In
analogous fashion, 0–30 mM of glucosamine 6-phosphate was
used as an inhibitor covering the same combinations and ranges of
substrate concentration as used in the experiments with NADPH.
The inhibition studies with compound C276-1187 [4-(4-
bromophenyl)-7-(3,4-dimethoxyphenyl)-3,4,7,8-tetrahydroqui-
noline-2,5(1H,6H)-dione; Chemdiv] were performed using 40–
300 μM C276-1187 both in the G6PD and the 6PGL assays.
Higher concentrations could not be used due to solubility
problems.
Gel-filtration chromatography was used to enhance the purity
of the protein samples as well as to study the oligomerization
behaviour of PfGluPho. The experiments were performed on a
HiLoad 16/60 Superdex 200 prep grade column connected to
¨
an AKTA FPLC system (Amersham Pharmacia Biotech). The
column was calibrated with a gel-filtration standard (Amersham
Pharmacia Biotech) and equilibrated with the respective buffer.
Protein-containing fractions were detected at 280 nm, and peak
areas and kAV values were evaluated using the software UNICORN
4.11. Protein-containing fractions were analysed by SDS/PAGE
(12 gel) and enzymatic assays. To study the oligomerization
behaviour, PfGluPho in 50 mM Tris/HCl and 0.3 M NaCl (pH 7.8)
was incubated for 12 h at 4◦C with 2 mM NADP+ , 2 mM
NADPH or 2 mM DTT (dithiothreitol) respectively. To study
the oligomerization behaviour at high pH and ionic strength,
PfGluPho was diluted in 0.5 M NaCl and 0.25 M Tris/HCl
(pH 9.0).
Measurement of kinetic parameters for the G6PD reaction
The G6PD activities of PfGluPho, PfGluPho’s G6PD part and
hG6PD were measured at 25◦C by monitoring the reduction
of NADP+ to NADPH at 340 nm using a method described
by Beutler [18]. The reaction mixture contained 0.1 M Tris/HCl
(pH 8.0), 10 mM MgCl2 and 0.5 mM EDTA with varying amounts
of enzyme, 200 μM NADP+ and 200 μM G6P (glucose 6-
phosphate), which was added to initiate the reaction. For the
determination of the Km for NADP+ the concentration of NADP+
was varied from 1–200 μM with 200 μM G6P, whereas the Km
for G6P was measured by varying the G6P concentration from
1–200 μM at 200 μM NADP+. The steady-state kinetics of the
G6PD activity for G6P were performed by fixing the concentration
of NADP+ at the Km, while varying the concentration of G6P (1–
200 μM). Likewise the steady-state kinetics for NADP+ were
performed by holding the concentration of G6P at a Km value
with different concentrations of NADP+ (1–200 μM). Enzyme
activity was assayed with a U-2001 spectrophotometer (Hitachi).
The kinetic parameters in the present study were calculated
by non-linear regression using the program GraphPad Prism, as
well as from Dalziel parameters. Dalziel’s method was applied
as an independent method to analyse and identify the kinetic
mechanism of PfGluPho, since it allows rival bisubstrate kinetic
mechanisms to be distinguished. The initial-rate equation for the
G6PD-catalysed reaction according to Dalziel is of the form (eqn
1) [19]:
Measurement of kinetic parameters for the 6PGL reaction
The 6PGL assays were carried out using the stable 6PGγ L
(6-phosphoglucono-γ -lactone) instead of the unstable natural
substrate 6PGδL (6-phosphoglucono-δ-lactone). 6PGγ L was
produced as described by Beutler et al. [20], and the 6PGL
activity was determined spectrophotometrically at 340 nm and
25◦C as described previously [20]. The assay mixture contained
0.1 M Tris/HCl (pH 7.0), 10 mM MgCl2, 0.5 mM EDTA, 0.6 mM
NADP+ , 3 units/ml 6-phosphogluconate dehydrogenase (yeast,
Sigma–Aldrich) and 1 mM 6PGγ L. For Km measurements, the
concentration of 6PGγ L was varied from 20–3000 μM.
S-glutathionylation studies
MALDI (matrix-assisted laser-desorption ionization)–TOF (time-of-flight)-MS
For investigating the susceptibility of PfGluPho to S-
glutathionylation, the recombinant enzyme (1 mg/ml) was
incubated with 10 mM GSSG in 50 mM Tris/HCl and 0.5 M NaCl
(pH 7.8) for 5 min at 37◦C. Iodoacetamide (2 mM) was added
to block residual cysteine residues. An analogous experiment
was performed for untreated PfGluPho. The samples were
digested with trypsin for 12 h at 37◦C. The tryptic peptides
were analysed by MALDI–TOF-MS on an Ultraflex I TOF/TOF
mass spectrometer (Bruker, Daltonics). A comparison of the
results with the theoretical molecular mass of the tryptic peptides
revealed cysteine residues with attached glutathione.
e
φS1
φS2
φS1S2
= φ0 +
+
+
(1)
v
[S1] [S2] [S1][S2]
where, S1 and S2 are the coenzyme (NADP+ ) and the sugar
phosphate (G6P) respectively, e is the total enzyme concentration,
ν represents the initial rate of the enzymatic reaction and ꢀ0
equals 1/kcat. The ꢀ parameters are calculated from initial-rate
measurements at varying concentrations of S2 for several fixed
Enzymatic assays
Enzymatic assays on S-glutathionylated G6PD were performed
after incubation of recombinant PfGluPho and hG6PD with
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Figure 1 SDS/PAGE and Western blot analyses of PfGluPho, hG6PD and h6PGL
(A) SDS/PAGE (12% gel) showing recombinant PfGluPho (6 μg). (B) Immunoblotting with anti-His6 antibody showing recombinant PfGluPho and hG6PD (5 μg). Immunoblotting with peptide
antibody against the C-terminal G6PD part of PfGluPho (anti-Glu) (C) and the N-terminal 6PGL part of PfGluPho (anti-Pho) (D) with recombinant PfGluPho (0.05 μg), h6PGL (5 μg) and hG6PD
(5 μg). Immunoblotting with anti-Glu (E) and anti-Pho (F) antibody with recombinant PfGluPho (0.05 μg) and P. falciparum lysate (100–200 μg). Molecular masses are shown on the left-hand side.
0–10 mM GSSG or GSH for 5 min at 37◦C. The reversibility
of the modification was studied by incubating PfGluPho with the
reducing agent DTT for 30 min at 23◦C after pre-incubation with
GSSG.
dimer and tetramer depending on ionic strength, pH and
the presence of its substrates [22]. According to the present
study, PfGluPho exists as a tetramer with a molecular mass
of 443 kDa under native conditions (Supplementary Figure S2
at http://www.BiochemJ.org/bj/436/bj4360641add.htm). The
tetrameric state is stable under reducing conditions (2 mM DTT),
indicating that the oligomerization is not based on intermolecular
disulfide bonds. Oligomerization studies after incubation with
NADP+ (2 mM) and NADPH (2 mM) revealed that PfGluPho
maintains its tetrameric structure in the presence of its substrate
and product inhibitor, as well as at alkaline pH.
RESULTS
Cloning, heterologous overexpression and purification of PfGluPho
Previous publications have reported major difficulties in cloning
PfGluPho, making it impossible to heterologously overexpress
and characterize the enzyme [9,10]. As reported in the present
paper, we were able to clone, overexpress and purify the
recombinant full-length PfGluPho protein (Figure 1). Cloning,
overexpression and purification of PfGluPho was extremely
challenging, dealing with major problems of insolubility, low
yield and degradation products. We used different E. coli strains
(KRX, Rosetta, C41, M15 and G6PD-deficient PD2000 cells),
combined with different helper plasmids (pRARE, pRARE II and
pRIG), a variety of growth, induction and expression conditions,
different purification strategies (Ni-NTA, 2ꢁ5ꢁ ADP-Sepharose 4B
and gel filtration), as well as several buffer systems. The best
condition yielded 2–3 mg of pure and active PfGluPho per litre of
E. coli culture. In addition to the full-length PfGluPho, we cloned
Western blot analyses
We obtained peptide-specific antibodies against the G6PD and
the 6PGL part of PfGluPho (anti-Glu and anti-Pho antibodies),
which are highly specific for PfGluPho since they do not react with
hG6PD or 6PGL (Figures 1C and 1D). In addition, we detected
full-length PfGluPho in P. falciparum parasite lysate using the
peptide antibodies (Figures 1E and 1F).
Besides the signal with a molecular mass of 107 kDa, the
expected molecular mass of recombinant PfGluPho deduced
from the amino acid sequence, we obtained
a second
signal of approximately 80 kDa. MALDI–TOF peptide-mass
fingerprinting of the two bands of recombinant PfGluPho
identified peptides from all parts of the full-length protein
[amino acids 9–911, coverage 55.8% (80 kDa band) and 48.6%
(107 kDa band)], indicating that both protein bands represent the
full-length protein and thus exclude a proteolytic truncation of
PfGluPho. The two bands were also detected with the peptide
antibodies in parasite lysate and thus occur in vivo (Figure 1). This
phenomenon might be due to post-translational modifications;
however, it remains to be studied in detail by high-accuracy MS.
the G6PD domain (amino acids 339–910, named G6PDPfGluPho
)
separately in order to test whether it functions independently of
the 6PGL part of the enzyme.
Oligomerization studies of PfGluPho
PfGluPho purified from parasite lysate has a tetrameric
structure [8,9,21], whereas hG6PD exists as a monomer,
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Plasmodium falciparum glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase
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Table 1 Kinetic characteristics of PfGluPho, G6PDPfGluPho, hG6PD and h6PGL
+
Each value is a mean S.D. from at least five independent determinations each including at least five measurements. nd, not determined. *Yoshida and Roth [7]; † O’Brien et al. [9]; ‡Wang et al.
−
[27]; §Birke et al. [23]; ꢂMiclet et al. [26].
Km (μM)
Vmax (units/mg)
PfGluPho’s G6PD activity
kcat (s− 1
)
Km (μM)
Vmax (units/mg)
Literature values
+
+
+
G6P
19.2 3.9
6.5 2.2
5.2 1.6
4.6 0.8
8.6 1.5
8.2 1.2
11*, 27†
0.8*, 4.5†
10000*
-
-
-
−
−
−
NADP+
2deoxyG6P
+
+
+
−
−
−
+
+
+
2670 320
4.1 0.8
7.3 1.5
−
−
−
G6PDPfGluPho
Literature values
Literature values
+
+
+
G6P
33.2 1.2
5.5 0.2
6.3 0.3
-
-
-
-
−
−
−
NADP+
6.1 0.7
5.5 0.1
6.3 0.1
+
+
+
−
−
−
Human G6PD
+
−
+
−
+
−
G6P
116 8.5
17.5 2.8
nd
64.4 11
57.7 15
nd
64.6 8.9
56.9 15
nd
54‡, 43§
6.7‡, 11§
1674‡
100‡, 170§
−
−
−
NADP+
2deoxyG6P
+
+
+
PfGluPho’s 6PGL activity
Literature values
Literature values
+
+
+
6PGγ L
6PGδL
172 36
nd
46.6 10
nd
106 24
nd
-
-
-
−
−
−
60ꢂ
Human 6PGL
+
+
+
6PGγ L
6PGδL
242 48
nd
1065 97
nd
505215 3083
nd
-
-
-
−
−
−
3330ꢂ
(Table 1 and 2). The data are displayed using linear transformation
to simplify interpretation of the results: the studies of PfGluPho’s
G6PD activity using different combinations of G6P and NADP+
yielded linear converging double reciprocal plots (Figure 2A).
An intersecting pattern was also observed with G6P as the
independent variable.
To study the G6PD part of PfGluPho independently from the
6PGL part, we also cloned and overexpressed the G6PD part
separately (G6PDPfGluPho). Interestingly G6PDPfGluPho catalyses the
reaction with the same specific activity as PfGluPho, but with
slightly higher Km values for both substrates (Table 1), indicating
that the G6PD activity is not largely influenced by the 6PGL part.
PfGluPho’s G6PD reaction
The G6PD activity of PfGluPho, as well as the activity of the
separately cloned G6PD part of PfGluPho (G6PDPfGluPho), were
characterized in detail and in direct comparison with hG6PD.
The kinetic studies were performed in the standard G6PD assay
buffer (0.1 M Tris/HCl, 10 mM MgCl2 and 0.5 mM EDTA,
pH 8.0) to allow a direct comparison with hG6PD from our
own studies as well as from previously published data. A pH
profile of the G6PD activity of PfGluPho in direct comparison
with hG6PD showed that both enzymes catalysed the reaction
with a maximum activity at pH 8.0 (Supplementary Figures S3A
and S3B at http://www.BiochemJ.org/bj/436/bj4360641add.htm).
EDTA stabilizes the dimeric form of hG6PD [23], but did not
influence the activity of PfGluPho (Figure S3C).
PfGluPho’s 6PGL reaction
hG6PD is stable for several months when stored at a
concentration of 30 mg/ml in 50 mM Tris/HCl, 300 mM NaCl
(pH 8.0) and 0.1 mM NADP+ at 4◦C. NADP+ binding to a
second ‘structural’ NADP+ -binding site is required for the long-
term structural integrity of the enzyme [24]. This phenomenon
is strongly conserved in eukaryotic organisms [25]. However,
NADP+ does not affect the stability or oligomerization state
of PfGluPho, suggesting that a putative second NADP+ -binding
site does not have a structural function in PfGluPho. PfGluPho
is stable for several months when stored at − 80◦C in 50 mM
Tris/HCl, 500 mM NaCl (pH 7.8) and 50% glycerol.
Studies on the activity of 6PGL are limited since the natural
substrate 6PGδL is highly unstable. Thus Beutler et al. [20]
optimized an assay system using the more stable γ -lactone
(6PGγ L) as a substrate for 6PGL, which contains a five-
membered heterocyclic ring, whereas the δ-lactone contains a
six-membered heterocyclic ring. We successfully employed
6PGγ L as a substrate to study PfGluPho in direct comparison
with h6PGL in an assay system coupled to the reaction of 6PGD,
the succeeding step of the PPP. PfGluPho catalyses the hydrolysis
of 6PGγ L with a specific activity of 46 units/mg (Table 1). Despite
the use of the synthetic 6PGγ L, this is a range comparable with an
NMR-based study where the 6PGL part of PfGluPho catalyses the
hydrolysis of 6PGδL with 60 units/mg [26]. The specific activity
of PfGluPho is remarkably lower when compared with h6PGL
with 1065 units/mg, although PfGluPho shows a slightly higher
substrate affinity (Table 1).
The G6PD activity of PfGluPho was directly compared
with hG6PD. The apparent Km of PfGluPho for NADP+ is
+
6.5 2.2 μM, while we calculated a Km of 8.1 μM from the
−
Dalziel parameters (according to Dalziel [19]). The apparent Km
+
for G6P is 19.2 3.9 μM, and 15.9 μM was calculated from the
−
Dalziel equations (Tables 1 and 2). The Km values differ slightly
due to differences in the methodology, such as concentration of
the cosubstrate. hG6PD has a comparatively lower affinity for
G6P and NADP+ , but a higher specific activity (Table 1).
The kinetic data were analysed by non-linear regression using
GraphPad Prism yielding kinetic constants and Dalziel parameters
Alternative substrate studies
Since alternative substrates can be used to differentiate kinetic
models, we used 2deoxyG6P as an alternative substrate for G6P
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Table 2 Dalziel parameters and ratios for the G6PD reaction of PfGluPho and hG6PD with the substrates NADP+ and G6P as well as 2deoxyG6P as an
alternative substrate
The results were determined from the primary plots against the reciprocal of NADP+ (row 1) and G6P (row 2). The mean value is shown in row (3). Values are represented as means S.D.
(A) PfGluPho with the substrates NADP+ and G6P
+
−
+
+
+
ꢀNADP
G6P
ꢀNADP
/
ꢀNADP
/
/
G6P
+
ꢀ0 (s)
ꢀNADP+ (μM*s)
ꢀG6P (μM*s)
ꢀNADP G6P (μM2*s)
ꢀNADP+G(6μP M)
ꢀG6P (μM)
ꢀG6PꢀNADP+ (s− 1
)
kcat (s− 1
)
+
−
+
−
+
−
+
−
1
2
3
0.16 0.01
0.17 0.02
0.16
1.27 0.17
1.36 0.13
1.31
2.49 0.11
2.67 0.38
2.58
20.2 2.29
21.6 2.22
20.9
15.9
15.9
15.9
8.1
8.1
8.1
6.4
5.9
6.2
6.4
5.9
6.2
−
−
−
−
+
+
+
+
(B) PfGluPho with the substrates NADP+ and 2deoxyG6P (2dG6P)
+
+
+
ꢀNADP
/
ꢀNADP
ꢀ2dG6P (μM)
/
ꢀNADP
/
2dG6P
2dG6P
+
ꢀ0 (s)
ꢀNADP+ (μM*s)
ꢀ2dG6P (μM*s)
ꢀNADP 2dG6P (μM2*s)
ꢀNADP+2(Gμ6PM)
ꢀ2dG6P ꢀNADP+ (s− 1
)
kcat (s− 1
)
)
+
+
+
+
1
2
3
0.19 0.01
0.82 0.19
425 32.6
1806 338
2200
2200
2200
4.25
4.25
4.25
5.2
4.2
4.69
5.18
4.2
4.69
−
−
−
−
+
+
+
+
0.24 0.05
0.22
1.01 0.44
0.92
524 77.9
474
2228 454
−
−
−
−
2017
(C) hG6PD with the substrates NADP+ and G6P
+
+
ꢀNADP+G6P
/
ꢀNADP
/
ꢀNADP
ꢀG6PꢀNADP+ (s− 1
/
G6P
G6P
+
ꢀ0 (s)
ꢀNADP+ (μM*s)
ꢀG6P (μM*s)
ꢀNADP G6P (μM2*s)
ꢀNADP+ (μM)
ꢀG6P (μM)
)
kcat (s− 1
+
−
+
−
+
−
+
−
1
2
3
0.02 0.002
0.02 0.004
0.02
0.28 0.05
0.34 0.09
0.31
1.77 0.45
2.15 0.11
1.96
19.9 0.47
23.5 0.92
21.7
111.9
111.9
111.9
17.3
17.3
17.3
55.0
59.0
57.5
55.0
59.9
57.5
−
−
−
−
+
+
+
+
substrate G6P than for the substrate analogue 2deoxyG6P. The
difference between natural and alternative substrate is emphasized
especially at low sugar phosphate concentrations, which is in
good agreement with the Dalziel parameters since ꢀ2deoxyG6P and
+
ꢀNADP
are much higher than those for G6P (Table 2).
2deoxyG6P
Inhibition studies
Inhibition studies with the product inhibitor NADPH and the
dead-end inhibitor glucosamine 6-phosphate were performed to
investigate the kinetic mechanism of PfGluPho’s G6PD activity
(Figure 3). Inhibition assays can be used to differentiate between a
sequential and a random-order reaction, and have been performed
for hG6PD and the inhibitors NADPH and glucosamine 6-
phosphate previously [27,28]. Glucosamine 6-phosphate is an
analogue of G6P and acts as a dead-end inhibitor, meaning that
the enzyme is completely inactive after binding of the inhibitor
[27,28]. NADPH was found to be a competitive inhibitor with
respect to NADP+ , as indicated by the intersection on the x-axis
in Figure 3(A) and increasing Km values with increasing inhibitor
concentrations (Figures 3C and 3D). Towards G6P, NADPH acts
as a mixed-type inhibitor (Figure 3B). Glucosamine 6-phosphate
acts as a mixed-type inhibitor with respect to NADP+ and shows
a competitive inhibition pattern towards G6P (Figures 3E and 3F),
which is sometimes a rather mixed type.
Analogous experiments with hG6PD demonstrate that NAPDH
acts as a competitive inhibitor towards NADP+ and non-
competitively with respect to G6P, whereas glucosamine 6-
phosphate showed a competitive inhibition towards G6P and a
non-competitive inhibition towards NADP+ .
Figure 2 PfGluPho’s double reciprocal plots for the reaction with NADP+
and G6P as substrates
(A) Primary plots of 1/v against 1/[NADP+ ] at various concentrations of G6P. (B) Secondary
plots of intercepts of primary plots against 1/[G6P]. (C) Secondary plots of slopes of primary
plots against 1/[G6P]. The experiments were performed three times and the reproducibility was
within 10%. The results of one representative experiment are shown.
The recently identified compound C276-1187 that has
antimalarial activity does bind to 6PGLPfGluPho [17], but we
could not detect an inhibitory effect on the 6PGL activity of
PfGluPho or h6PGL at concentrations up to 300 μM. However,
the compound was found to inhibit the G6PD activity of
in the G6PD reaction as shown in Supplementary Figure S4
(at http://www.BiochemJ.org/bj/436/bj4360641add.htm) and
Table 1. As expected, PfGluPho has a higher affinity for its natural
+
PfGluPho with an IC50 of 127 17 μM. Compound C276-
−
1187 acts on PfGluPho’s G6PD activity as a non-competitive
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Figure 3 Inhibition studies of PfGluPho by the product inhibitor NADPH (A–D) and the dead-end inhibitor glucosamine 6-phosphate (E–H) with varied NADP+
and varied G6P concentrations
Lineweaver–Burk plots were obtained at 60 μM G6P and different NADP+ concentrations (A and E) or at 10 μM NADP+ and different G6P concentrations (B and F) both in the presence of
0–30 μM NADPH or 0–30 mM glucosamine 6-phosphate respectively. The apparent Km values for NADP+ (C and G) and G6P (D and H) are plotted against the concentration of NADPH or
glucosamine 6-phosphate. Each experiment was performed three times and the reproducibility was within 15%. The results of one representative experiment are shown.
inhibitor towards both G6P and NADP+ (Figure S5 at http://www.
BiochemJ.org/bj/436/bj4360641add.htm). G6PDPfGluPho is inhib-
regulated by S-glutathionylation in a concentration-dependent
manner (Figures 4A and 4C). In contrast, incubation with GSH
as well as DTT increases PfGluPho’s G6PD activity by 25%,
whereas the 6PGL activity is not affected by reduction. Incubation
of the S-glutathionylated PfGluPho with DTT showed that the
inhibition of both activities could be partially reversed (∼80%)
by DTT, showing that PfGluPho is inhibited by a reversible
mechanism (Figures 4B and 4D). In contrast, hG6PD was not
found to be regulated by GSSG, whereas h6PGL showed a similar
inhibition by S-glutathionylation when compared with PfGluPho
(Figures 4B and 4C).
+
ited with an IC50 of 130 11 μM, whereas the inhibition of
−
+
hG6PD is more efficient with an IC50 of 76 16 μM.
−
S-Glutathionylation studies on PfGluPho
A previous study suggests that PfGluPho contains a peptide
with high similarity to the glutathione-binding site of
glutathione transferase [9]. This motivated us to test whether
recombinant PfGluPho is S-glutathionylated using MALDI–TOF-
MS and kinetic analyses. Recombinant PfGluPho incubated with
glutathione disulfide (GSSG) was analysed by peptide-mass
fingerprinting with MALDI–TOF after trypsin digestion. The
matched peptides covered 38% of the protein. A clear mass
DISCUSSION
For the first time we describe the cloning, heterologous
overexpression, purification and kinetic characterization of
PfGluPho, a bifunctional enzyme combining G6PD and 6PGL
of the PPP from P. falciparum with unique structural and
functional characteristics. The present study demonstrates that
PfGluPho shows remarkable differences to the corresponding
human enzymes regarding the primary structure, oligomerization
behaviour, substrate affinity and regulation, as well as the kinetic
mechanism.
increase of ∼305 Da could be shown for the peptides Ser140
Cys144, Cys507, Cys806 and Cys838 respectively. Except for Cys144
–
Lys151, Asn506–Arg524, Thr804–Lys821 and Lys824–Lys840 containing
,
all S-glutathionylated cysteine residues are located in the G6PD
domain.
The G6PD assay was performed after incubating PfGluPho
with GSSG or reduced glutathione (GSH) and demonstrated that
PfGluPho’s G6PD activity, as well as its 6PGL activity, are down-
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Figure 4 S-Glutathionylation of PfGluPho and hG6PD
◦
Concentration-dependent inactivation of PfGluPho’s G6PD (A) and PfGluPho’s 6PGL (C) activity after incubation with different GSSG concentrations for 5 min at 37 C. Activity is given as percentage
of initial activity. (B) Activity of PfGluPho’s G6PD (grey bars) and hG6PD (black bars) after incubation with glutathione. (D) Activity of PfGluPho’s 6PGL (grey bars) and h6PGL (black bars) after
+
incubation with glutathione. Each value is a mean S.D. from at least three independent determinations each including five measurements.
−
Previous attempts to clone the full-length PfGluPho failed, most
probably due to the high AT content and the size of the gene
[10]. Cloning and overexpression of the 6PGL part of PfGluPho
yielded only 10 μg of protein per litre of E. coli culture, since most
of the protein was insoluble [26]. Thus studies using PfGluPho
have so far been restricted to the enzyme purified from parasite
extract [7,8,21], which is limiting in terms of the enzyme quantity
required for biochemical characterization, inhibitor tests and
crystallization screening, and might be contaminated with G6PD
from human RBCs. Cloning, overexpression and purification of
full-length PfGluPho was challenging, but finally we were able to
produce 2–3 mg of pure PfGluPho per litre of E. coli culture.
According to our studies, recombinant PfGluPho is a tetramer
of 443 kDa (Supplementary Figure S2), which is in agreement
with the tetrameric structure of PfGluPho purified from parasite
extract [8,9,21]. h6PGL is described as a monomer [29], whereas
hG6PD exists in transition between dimer and tetramer depending
on ionic strength, pH and substrate concentration, with the dimer
probably being the predominant form in vivo [23,30]. In contrast,
the quaternary structure of PfGluPho is not influenced by the
presence of NADP+ , DTT or NADPH, and is also stable at
alkaline pH. A dimeric or monomeric form could not be detected
in our experiments.
the results of the present study. Nevertheless our Km values
for the G6PD activity of PfGluPho obtained from the pure
recombinant enzyme are similar to those reported by Kurdi–
Haidar and Luzzatto (Table 1) [8]. Analysis of the separately
cloned G6PD part of PfGluPho (G6PDPfGluPho) demonstrates that
the fusion of G6PD and 6PGL does not enhance the efficiency of
the first PPP reaction.
PfGluPho has higher substrate affinities for both NADP+
and G6P. This implies that in infected RBCs, where G6P and
particularly NADP+ concentrations are lower than the Km values
of hG6PD, PfGluPho utilizes most of the G6P and NADP+
present for the generation of NADPH, although this effect might
in part be compensated by the higher specific activity of hG6PD
(63.9 units/mg compared with 4.5 units/mg).
The kinetic mechanism of hG6PD has been controversially
discussed by several groups, which might be due to the
heterogeneous origin of hG6PD used in the respective studies
[23,28]. The discrepancy was solved by Wang et al. [27] who
clearly documented a rapid equilibrium random-order mechanism
for recombinant hG6PD. We applied product inhibition studies
to elucidate the binding order of substrates and release order
of products as an independent test to differentiate between an
ordered and a random mechanism. The studies of PfGluPho’s
G6PD activity point to a rapid equilibrium random Bi Bi system,
in which both substrates must bind before product formation can
occur. In both cases the binding of the second substrate depends
on the binding of the first substrate and indicates a sequential
order mechanism of substrate binding; however, without a leading
PfGluPho exhibits both G6PD and 6PGL activity, which we
characterized in detail for the first time using the recombinant
full-length enzyme. Previous kinetic data on the G6PD activity of
PfGluPho were obtained from parasite extracts, which explains
the small discrepancies observed between previous studies and
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Plasmodium falciparum glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase
649
Figure 5 PfGluPho’s G6PD reaction follows a rapid equilibrium random Bi Bi mechanism
The binding of the second substrate depends on the binding of the first substrate. Both substrates must bind to PfGluPho before product formation can occur.
a low NADP+/NADPH ratio by avoiding that the uncatalysed
hydrolysis becomes rate-limiting for the dehydrogenase reactions
of the PPP [29]. Regarding the dependence of the malaria parasite
on the PPP and the tight regulation, the combination of G6PD
and 6PGL in PfGluPho might improve the efficiency and the
regulation of the PPP.
substrate in PfGluPho. Thus in the parasite enzyme it is not
important which substrate binds first, but the two substrates cannot
bind simultaneously. Analogous experiments with hG6PD imply
that hG6PD acts via a rapid-equilibrium random-order mechanism
with both binding sites acting independently from each other,
which has been proposed by Wang et al. [27] previously.
An independent examination of the kinetic mechanism was
applied using the Dalziel parameters to test whether PfGluPho’s
G6PD activity acts according to a compulsory-order mechanism
where one substrate is the leading substrate. If substrate 1
A recent chemical genetic screen identified two compounds that
are active against drug-resistant P. falciparum strains and bind to
6PGLPfGluPho in thermal melt-shift experiments [17]. We were able
to obtain one of the two compounds (C276-1187) and could show
that it does not inhibit the 6PGL activity of PfGluPho, but acts as a
non-competitive inhibitor towards the G6PD activity of PfGluPho.
The weak inhibition of PfGluPho by C276-1187 suggests that its
antimalarial activity is not mainly due to PfGluPho inhibition.
Furthermore, the compound is not specific for PfGluPho, since it
is more active on hG6PD.
(NADP+) binds first to the enzyme, ꢀNADP G6P/ꢀG6P is Kd,
+
the dissociation constant for substrate 1 leaving the binary-
+
enzyme complex. This value as well as the values for ꢀNADP
+
+
and ꢀNADP G6P/ꢀG6PꢀNADP should not change if an alternative
substrate 2 is used, whereas the values of ꢀ0 (1/kcat), ꢀG6P
can be different [19]. Whether NADP+ is the
+ G6P
and ꢀNADP
leading substrate can be deduced from parameters that were
Protein S-glutathionylation is a reversible process that
regulates the activity of several proteins via functionally or
structurally critical cysteine residues (reviewed in [31]). MS
and enzymatic data clearly demonstrate that PfGluPho can
undergo S-glutathionylation, which leads to a concentration-
dependent and reversible inactivation of both the G6PD and
the 6PGL activity (Figure 4), indicating that the intracellular
GSH/GSSG ratio regulates PfGluPho activity. hG6PD activity
did not respond to glutathione, whereas h6PGL was affected
by high glutathione concentrations (Figures 4B and 4D). A
comparable glutathione-mediated inhibition has been shown for
h6PGL previously [32]. PfGluPho is completely inhibited by
8 mM GSSG, a concentration that might occur locally in the cell
under high oxidative stress. This suggests that S-glutathionylation
of PfGluPho might rather be important for protection of PfGluPho
from irreversible oxidation under high oxidative stress than for
regulation of enzyme activity.
The PPP of human erythrocytes is highly up-regulated
after Plasmodium infection, with the PPP of malaria parasites
accounting for approximately 80% of the total PPP activity in
infected erythrocytes. This up-regulation is attributed to high
oxidative stress in infected erythrocytes, since the PPP provides
NADPH for reducing reactions. Additionally, a high turnover in
the parasitic PPP resulting in high concentrations of ribose 5-
phosphate is required for the synthesis of nucleotides [6]. G6PD
has been shown to be the rate-limiting enzyme in the PPP, and is
essential for the survival of various organisms such as mice [33]
and trypanosomes [34]. Transient silencing of PfGluPho results in
growth arrest at the trophozoite stage, suggesting that PfGluPho
is crucial for malaria parasites [13]. PfGluPho as the first and
presumably rate-limiting enzyme of the Plasmodium PPP must
be tightly regulated, an aspect that has to be addressed in future
studies.
obtained using 2deoxyG6P instead of G6P (Table 2): if NADP+
+
is the leading substrate, ꢀNADP G6P/ꢀG6P should be equal to
+
ꢀNADP 2deoxyG6P/ꢀ2deoxyG6P. We obtained with 8.1 μM and 4.3 μM
+
quite different values for these parameters. ꢀNADP with G6P
+
as a substrate was 1.3 μM · s, while ꢀNADP with 2deoxyG6P
as a substrate was 0.9 μM · s. In parallel, the mean values
for ꢀNADP G6P/ꢀG6PꢀNADP are with 6.2 s− 1 (G6P) and 4.7 s− 1
(2deoxyG6P) different. These results exclude a compulsory-order
mechanism with NADP+ as the leading substrate for PfGluPho
and support the results of the inhibition studies.
+
+
Product inhibition studies and an alternative substrate imply
that the reaction of PfGluPho’s G6PD activity follows a rapid
equilibrium random Bi Bi system without a leading substrate
where both substrates cannot bind simultaneously (Figure 5).
This kinetic mechanism clearly distinguishes PfGluPho from
hG6PD, which follows a rapid equilibrium random-order
mechanism with both substrates binding independently from each
other [27].
In addition to the G6PD activity we examined PfGluPho’s
6PGL activity for the first time. Owing to instability of the
substrate, the enzymatic properties of 6PGL from various
organisms including humans are barely studied. Using the γ -
lactone instead of the unstable δ-lactone as a substrate to study
PfGluPho’s 6PGL activity, we did not only confirm the specific
activity using 6PGδL in a previous NMR-based study (Table 1),
but also established a reproducible and reliable system to compare
PfGluPho and h6PGL in future inhibitor screens. A previous
study reported that the γ -lactone can neither spontaneously nor
enzymatically be hydrolysed and forms a ‘dead-end’ of the PPP
[26]. However, according to the present study, both PfGluPho
and h6PGL accept the γ -lactone as a substrate. Compared with
the human enzyme, PfGluPho’s 6PGL activity is rather low
(Table 1). The relevance of 6PGL was questioned, since its
substrate 6PGδL undergoes spontaneous hydrolysis. However,
enzyme-catalysed hydrolysis of 6PGδL is required to maintain
The present study revealed major differences between hG6PD
and PfGluPho: the bifunctional enzyme is not expressed in
mammals, contains an insertion sequence with unknown but
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12 Clarke, J. L., Sodeinde, O. and Mason, P. J. (2003) A unique insertion in Plasmodium
berghei glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: evolutionary
and functional studies. Mol. Biochem. Parasitol. 127, 1–8
13 Crooke, A., Diez, A., Mason, P. J. and Bautista, J. M. (2006) Transient silencing of
Plasmodium falciparum bifunctional glucose-6-phosphate dehydrogenase –
6-phosphogluconolactonase. FEBS J. 273, 1537–1546
essential function, has a stable tetrameric structure, is inhibited
by S-glutathionylation and differs in substrate affinity as well
as kinetic mechanism from the human counterparts. The unique
characteristics of PfGluPho provide the starting point for
developing new antimalarial drugs.
14 Cerutti, H. and Casas-Mollano, J. A. (2006) On the origin and functions of RNA-mediated
silencing: from protists to man. Curr. Genet. 50, 81–99
15 Xue, X., Zhang, Q., Huang, Y., Feng, L. and Pan, W. (2008) No miRNA were found in
Plasmodium and the ones identified in erythrocytes could not be correlated with infection.
Malar. J. 7, 47
AUTHOR CONTRIBUTION
Esther Jortzik, Boniface Mailu, Janina Preuss, Lars Bode, Stefan Rahlfs and Katja Becker
conceived and designed the experiments. Esther Jortzik, Boniface Mailu, Janina Preuss
and Marina Fischer carried out the experiments. Esther Jortzik, Stefan Rahlfs and Katja
Becker analysed the data. Esther Jortzik and Katja Becker wrote the manuscript.
16 Rahlfs, S. and Becker, K. (2006) Interference with redox-active enzymes as a basis for the
design of antimalarial drugs. Mini Rev. Med. Chem. 6, 163–176
17 Guiguemde, W. A., Shelat, A. A., Bouck, D., Duffy, S., Crowther, G. J., Davis, P. H.,
Smithson, D. C., Connelly, M., Clark, J., Zhu, F. et al. (2010) Chemical genetics of
Plasmodium falciparum. Nature 465, 311–315
18 Beutler, E. (1984) Red cell metabolism: a manual of biochemical methods, Grune and
Stratton, New York
19 Dalziel, K. (1957) Initial steady state velocities in the evaluation of enzyme-coenzyme-
substrate reaction mechanisms. Acta Chem. Scand. 11, 1706–1723
20 Beutler, E., Kuhl, W. and Gelbart, T. (1986) Blood cell phosphogluconolactonase: assay
and properties. Br. J. Haematol. 62, 577–586
ACKNOWLEDGEMENTS
The authors would like to thank Professor Charles William Jr, Professor David Ballou
and Professor David Arscott (University of Michigan, Ann Arbor, MI, U.S.A.), for helpful
discussions on the kinetic mechanism of PfGluPho. We would also like to thank Michaela
Stumpf and Beate Hecker for their excellent technical assistance.
21 Ling, I. T. and Wilson, R. J. (1988) Glucose-6-phosphate dehydrogenase activity of the
malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 31, 47–56
22 Wrigley, N. G., Heather, J. V., Bonsignore, A. and De Flora, A. (1972) Human erythrocyte
glucose 6-phosphate dehydrogenase: electron microscope studies on structure and
interconversion of tetramers, dimers and monomers. J. Mol. Biol. 68,
483–499
FUNDING
The study was supported by the Deutsche Forschungsgemeinschaft [grant number
BE1540/15-1] and the National Institutes of Health [grant number 1R21AI082434-01].
23 Birke, S., Kim, H. W., Periclou, A., Schorsch, B., Grouse, D. and Craney, C. (1989)
Kinetics of human erythrocyte glucose-6-phosphate dehydrogenase dimers. Biochim.
Biophys. Acta 999, 243–247
24 Wang, X. T., Chan, T. F., Lam, V. M. and Engel, P. C. (2008) What is the role of the second
‘structural’ NADP + -binding site in human glucose 6-phosphate dehydrogenase? Protein
Sci. 17, 1403–1411
25 Au, S. W., Gover, S., Lam, V. M. and Adams, M. J. (2000) Human glucose-6-phosphate
dehydrogenase: the crystal structure reveals a structural NADP( + ) molecule and
provides insights into enzyme deficiency. Structure 8, 293–303
26 Miclet, E., Stoven, V., Michels, P. A., Opperdoes, F. R., Lallemand, J. Y. and Duffieux, F.
(2001) NMR spectroscopic analysis of the first two steps of the pentose-phosphate
pathway elucidates the role of 6-phosphogluconolactonase. J. Biol. Chem. 276,
34840–34846
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Received 25 January 2011/14 March 2011; accepted 28 March 2011
Published as BJ Immediate Publication 28 March 2011, doi:10.1042/BJ20110170
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Biochem. J. (2011) 436, 641–650 (Printed in Great Britain) doi:10.1042/BJ20110170
SUPPLEMENTARY ONLINE DATA
Glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase: a
unique bifunctional enzyme from Plasmodium falciparum
Esther JORTZIK*1, Boniface M. MAILU*1, Janina PREUSS*†, Marina FISCHER*, Lars BODE†, Stefan RAHLFS* and
Katja BECKER*2
*Interdisciplinary Research Center, Justus Liebig University, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany, and †Department of Pediatrics, University of California San Diego,
200 West Arbor Drive, San Diego, CA 92103-8450, U.S.A.
Figure S1 Schematic presentation of the PfGluPho gene and protein
aa, amino acid.
Figure S2 Oligomerization of PfGluPho
Under native conditions, PfGluPho elutes from the gel-filtration column as a tetramer with an
apparent molecular mass of 443 kDa.
1
These authors contributed equally to this work.
To whom correspondence should be addressed (email katja.becker@ernaehrung.uni-giessen.de).
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E. Jortzik and others
Figure S3 pH profile of hG6PD (A) and the G6PD activity of PfGluPho (B), as well as the effect of EDTA on the G6PD activity of PfGluPho (C)
◦
(A) and (B) The G6PD activity was determined in the presence of 200 μM NADP+ and 200 μM G6P in 0.1 mM Tris/HCl, 10 mM MgCl2 and 0.5 mM EDTA at 25 C. The pH was varied in steps of
◦
0.2. (C) PfGluPho’s G6PD activity was measured in the presence of 200 μM NADP+ and 200 μM G6P in 0.1 mM Tris/HCl and 10 mM MgCl2 (pH 8.0) at 25 C either with 0 mM (dark grey bars) or
+
0.5 mM (light grey bars) EDTA. Each value is a mean S.D. from at least three independent determinations each including five measurements.
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Plasmodium falciparum glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase
Figure S4 PfGluPho’s double reciprocal plots for the reaction with NADP+
and the alternative substrate 2-deoxy-glucose 6-phosphate (2deoxyG6P) as
substrates
(A) Primary plots of 1/v against 1/[NADP+ ] at various concentrations of 2deoxyG6P. (B)
Secondary plots of intercepts of primary plots against 1/[2deoxyG6P]. (C) Secondary plots of
slopes of primary plots against 1/[2deoxyG6P].
Figure S5 Inhibition of PfGluPho by compound C276-1187
Lineweaver–Burk plots were obtained at 20 μM NADP+ and different G6P concentrations
(A) or at 60 μM G6P and different NADP+ concentrations (B) in the presence of different
concentrations of C276-1187. The apparent Km values for NADP+ (C) and G6P (D) are plotted
against the concentration of the compound C276-1187.
Received 25 January 2011/14 March 2011; accepted 28 March 2011
Published as BJ Immediate Publication 28 March 2011, doi:10.1042/BJ20110170
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