Thus, whereas the structural features of PfFNR and PfFd are now known in great detail, the functional properties of the protein couple have been poorly analyzed.. Results PfFNR gene clon
Trang 1transfer system of Plasmodium falciparum
Emanuela Balconi, Andrea Pennati*, Danila Crobu, Vittorio Pandini, Raffaele Cerutti,
Giuliana Zanetti and Alessandro Aliverti
Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita` degli Studi di Milano, Italy
Keywords
disulfide; enzyme; flavoprotein; hydride
transfer; malaria
Correspondence
A Aliverti, Dipartimento di Scienze
Biomolecolari e Biotecnologie, Universita`
degli Studi di Milano, via Celoria 26, 20133
Milano, Italy
Fax: +39 02 50314895
Tel: +39 02 50314897
E-mail: alessandro.aliverti@unimi.it
Website: http://www.bsb.unimi.it/
*Present address
Department of Chemistry, Georgia State
University, Atlanta, GA, USA
(Received 18 March 2009, revised 30 April
2009, accepted 13 May 2009)
doi:10.1111/j.1742-4658.2009.07100.x
In the apicoplast of apicomplexan parasites, plastidic-type ferredoxin and ferredoxin-NADP+reductase (FNR) form a short electron transport chain that provides reducing power for the synthesis of isoprenoid precursors These proteins are attractive targets for the development of novel drugs against diseases such as malaria, toxoplasmosis, and coccidiosis We have obtained ferredoxin and FNR of both Toxoplasma gondii and Plasmo-dium falciparum in recombinant form, and recently we solved the crystal structure of the P falciparum reductase Here we report on the functional properties of the latter enzyme, which differ markedly from those of homolo-gous FNRs In the physiological reaction, P falciparum FNR displays a kcat five-fold lower than those usually determined for plastidic-type FNRs By rapid kinetics, we found that hydride transfer between NADPH and protein-bound FAD is slower in the P falciparum enzyme The redox properties of the enzyme were determined, and showed that the FAD semiquinone species
is highly destabilized We propose that these two features, i.e slow hydride transfer and unstable FAD semiquinone, are responsible for the poor cata-lytic efficiency of the P falciparum enzyme Another unprecedented feature
of the malarial parasite FNR is its ability to yield, under oxidizing condi-tions, an inactive dimeric form stabilized by an intermolecular disulfide bond Here we show that the monomer–dimer interconversion can be controlled by oxidizing and reducing agents that are possibly present within the apicoplast, such as H2O2, glutathione, and lipoate This finding suggests that modulation
of the quaternary structure of P falciparum FNR might represent a regula-tory mechanism, although this needs to be verified in vivo
Structured digital abstract
l MINT-7042675 : PfFNR (genbank_protein_gi: 46361180 ) and PfFNR (genbank_protein_ gi: 46361180 ) bind ( MI:0408 ) by comigration in sds page ( MI:0808 )
l MINT-7043503 : PfFNR (genbank_protein_gi: 46361180 ) and PfFNR (genbank_protein_ gi: 46361180 ) bind ( MI:0408 ) by molecular sieving ( MI:0071 )
l MINT-7043886 , MINT-7051941 : PfFd (uniprotkb: Q8IED5 ) and PfFNR (genbank_protein_ gi: 46361180 ) enzymaticly react ( MI:0414 ) by enzymatic studies ( MI:0415 )
l MINT-7051926 : PfFNR (genbank_protein_gi: 46361180 ) and PfFd (uniprotkb: Q8IED5 ) enzymaticly react ( MI:0414 ) by biophysical ( MI:0013 )
Abbreviations
CT1, charge transfer complex between NADPH and enzyme-bound FAD; CT2, charge transfer complex between NADP + and fully reduced enzyme-bound FAD; EDC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; Fd, ferredoxin; FNR, ferredoxin-NADP + reductase; GSH, glutathione; IPTG, isopropyl thio-b- D -galactoside; INT, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride; PfFd,
Plasmodium falciparum ferredoxin; PfFNR, Plasmodium falciparum ferredoxin-NADP + reductase.
Trang 2The protozoan parasite Plasmodium falciparum causes
more than 1 000 000 deaths every year [1] The
devel-opment of resistance in the parasite to the presently
used drugs means that new targets for novel
antimalar-ial drugs need to be developed Plasmodia, like all of
the Apicomplexa except for Cryptosporidium spp.,
contain a unique organelle, the apicoplast, that is
phylogenetically and functionally related to plant
nonphotosynthetic plastids [2,3] The apicoplast has
been shown to be a major determinant of virulence [4],
strongly suggesting that proteins located therein could
provide new and promising targets for rational drug
design, because it is assumed to contain a number
of unique metabolic pathways not found in the
mammalian host Recently, it has been reported that
Toxoplasma gondii and P falciparum possess a
nuclear-encoded but apicoplast-localized redox system
comprising a plastidic-type ferredoxin-NADP+
reduc-tase (FNR; EC 1.18.1.2) and a [2Fe–2S] ferredoxin
(Fd) [5,6] All other apicomplexan parasites examined
so far also possess DNA sequences for
apicoplast-targeted Fd and FNR, implying that this redox system
is of primary importance for the parasite Although
the physiological role of the FNR⁄ Fd couple in the
apicomplexan parasites is still largely unknown, it is
conceivable that it could provide reducing power for
various reductive biosyntheses Indeed, we have
recently demonstrated that, in vitro, the FNR⁄ Fd
system of P falciparum can supply electrons to LytB,
an enzyme of the apicoplast isoprenoid biosynthetic
pathway [7], which is a known target of antiplasmodial
compounds [8]
In the past, we produced both the FNR and Fd of
T gondii in recombinant form, and characterized this
electron transport system in detail [9–12]
Unfortu-nately, we did not succeed in obtaining crystals of
these proteins that were suitable for X-ray analysis
With the aim of obtaining new insights into the
struc-ture–function relationships of the apicomplexan
FNR⁄ Fd system, we then accomplished the
heterolo-gous expression in Escherichia coli of the P falciparum
FNR (PfFNR) gene Crystals of the recombinant
reductase were obtained, and its three-dimensional
structure was solved in free form and complexed with
the inhibitor 2¢-P-AMP at 2.4 and 2.7 A˚ resolution,
respectively [13] An unexpected feature, peculiar to
PfFNR, was the finding that it crystallizes as a dimer
stabilized by an intermolecular disulfide bridge linking
the Cys99 side chains of each protomer We
demon-strated that PfFNR dimerization in solution occurs
under oxidizing conditions and is highly favored by
2¢-P-AMP as well as NADP+ binding The covalent dimer is essentially inactive, and disulfide reduction restores full activity [13] More recently, the three-dimensional structure of P falciparum Fd (PfFd) and the identification of its surface regions involved in the interaction with the reductase have been reported [14,15] Thus, whereas the structural features of PfFNR and PfFd are now known in great detail, the functional properties of the protein couple have been poorly analyzed In order to fill this gap, we report here on the distinctive functional properties of the
P falciparum FNR⁄ Fd redox system in comparison with other known FNR⁄ Fd couples The new informa-tion that we provide here is expected to increase our knowledge of the structure–function relationships of PfFNR, which could be helpful for the rational design
of PfFNR⁄ PfFd inhibitors, possibly endowed with antimalarial activity
Results
PfFNR gene cloning and heterologous expression Using plasmid pET–PfFNR, the putative mature PfFNR, starting at Lys56 of the deduced sequence (the first amino acid of the mature form was chosen
on the basis of sequence alignments with FNRs where the boundary between the signal N-terminal sequence and mature polypeptide is known), was overproduced
in E coli as a fusion protein with an N-terminal His-tag extension A factor Xa recognition site, engineered between the poly-His and the PfFNR sequence, allowed tag removal during protein purification Not-withstanding the high A⁄ T content of its coding sequence, a substantial production of PfFNR was obtained in E coli using as host the Rosetta(DE3) strain, which is specifically engineered to enhance the expression of eukaryotic proteins containing codons rarely used in E coli Optimal production of the enzyme was obtained by growing transformed cells at
20C for 24 h after isopropyl thio-b-d-galactoside (IPTG) induction The purification procedure included
a step on a phenyl–Sepharose column after the metal chelate affinity chromatography Following the factor
Xa proteolysis step, a second affinity column step was performed, which allowed the removal of both small amounts of undigested enzyme and contaminants, exploiting weak binding affinity of the untagged PfFNR for the Ni2+–Sepharose resin About 1.5 mgÆg)1cells of homogeneous enzyme were obtained, with a 26% over-all yield Recombinant PfFd was difficult to produce in
Trang 3substantial amounts with the previously described
expression plasmid [5], so the DNA fragment encoding
the putative mature form of PfFd, starting at Leu97,
was transferred to plasmid pET28b without the
addi-tion of any affinity tag A three-fold improvement of
holo-PfFd yield was obtained by cotransforming the
E colicells with a plasmid carrying the isc gene cluster
to favor iron–sulfur biogenesis About 2.5 mg of
puri-fied PfFd were obtained from 1 g of cells with a 26%
yield PfFd showed the typical spectrum of a 2Fe Fd,
with peaks at 277, 424 and 460 nm, and a value of 0.59
for the A424 nm⁄ A280 nmratio
Steady-state kinetic analyses of PfFNR
The crystal structure of the 2¢-P-AMP–PfFNR
com-plex suggested that the His286 side chain needs to be
charged to effectively bind the substrate [13] In order
to verify this hypothesis, we determined at pH 7.0 the
kinetic parameters of the diaphorase reactions of
PfFNR, previously measured at pH 8.2 [13] Table 1
shows that there is, indeed, a decrease in the Kmvalues
for NADPH with both ferricyanide and
2-(4-iodo-phenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride
(INT) as electron acceptors, by approximately
four-fold and seven-four-fold, respectively, whereas the kcat
val-ues were only moderately lowered (by 30%) Thus,
PfFNR shows a three-fold to five-fold higher catalytic
efficiency as a diaphorase at pH 7.0 than at pH 8.2
[13], which is at variance with the plastidic-type FNRs
The physiological reaction of PfFNR was initially
measured with recombinant T gondii Fd, which was
available in our laboratory The kcat value was only
20% of that obtained with the homologous couple of
T gondii, and the Km value for T gondii Fd was
two-fold higher This poor catalytic activity prompted us to produce PfFd in recombinant form The kinetic parame-ters of the physiological reaction of PfFNR with PfFd were determined at both pH 8.2 and pH 7.0 (Table 2)
At pH 8.2, the Kmof PfFNR for the homologous pro-tein substrate was indeed in the same range as those of other plastidic-type FNR⁄ Fd couples The kcat values with both apicomplexan Fds were similar, and about five-fold lower than those of plastidic-type FNRs [9,16,17] As expected, at pH 7.0, the Kmfor NADPH was markedly diminished (11-fold lower than that at pH 8.2), whereas the kcat was halved The three-fold decrease in the Kmvalue for PfFd on lowering of the pH from 8.2 to 7.0 would suggest stronger binding also of the protein substrate at neutral pH Thus, also in the physiological reaction, PfFNR shows a higher catalytic efficiency at pH 7 than at pH 8.2
PfFNR covalent dimer The covalent dimer of PfFNR, which was shown to maintain only 5% of the kcat value in the NADPH– ferricyanide reaction [13], was found to be fully inactive
in catalysing the physiological NADPH–cytochrome c reductase reaction (PfFd-dependent) We further inves-tigated the conditions that promote Cys99 disulfide for-mation to give the homodimer, and tested the ability of physiological reductants to reverse the dimerization and re-establish the catalytic activity of PfFNR For easy analysis of the monomer–dimer interconversion under different conditions, the procedure reported by Leichert and Jacob [18] was followed Briefly, after the desired treatment was performed, N-ethylmaleimide and SDS were added to the samples in order to block all protein-free sulfhydryls SDS⁄ PAGE was then
per-Table 1 Kinetic parameters for the diaphorase reactions of PfFNR Activity assays were performed at 25 C The kinetic parameters at pH 8.2 were taken from [13].
pH Reaction kcat(e)eqÆs)1) KmNADPH (l M ) kcat⁄ K mNADPH(e)eqÆs)1Æl M )1)
Table 2 Kinetic parameters for the cytochrome c reductase (PfFd-dependent) reaction of PfFNR Activity assays were performed at 25 C.
pH kcat(e)eqÆs)1) KmNADPH (l M ) kcat⁄ K mNADPH(e)eqÆs)1Æl M )1) K
mPfFd(l M ) kcat⁄ K mPfFd(e)eqÆs)1Æl M )1)
Trang 4formed under nonreducing conditions, in order to
pre-serve the disulfide bonds possibly present in the
pro-tein Incubation of PfFNR with different
concentrations of hydrogen peroxide in the presence or
absence of NADP+ showed that this reactive oxygen
species is far more efficient than O2 [13] in promoting
formation of the enzyme dimer, which accumulated in
significant amounts only when the ligand was present
in the incubation mixture (Fig 1A,B) The time course
of the decrease of the enzyme diaphorase activity
matched the formation of the dimer (Fig 1C)
Oxi-dized lipoic acid was also able to induce enzyme
dimer-ization, although at a lower rate: 40% dimer was
formed in 3 h in the presence of 100 lm lipoic acid and
1 mm NADP+ The PfFNR dimer purified by gel
fil-tration was converted back to the monomer by
incuba-tion with dithiothreitol [13], glutathione (GSH), or
dihydrolipoate The time course of production of
monomer by incubation of 7.5 lm PfFNR dimer with
50 lm dihydrolipoate was determined both by
nonreducing SDS⁄ PAGE (Fig 2A) and by measuring
the diaphorase activity (Fig 2B) The half-life for
conversion was 7 min In similar experiments, 100 lm
GSH required more time for full conversion (half-life
of 11 min)
With the aim of studying the role of Cys99 of
PfFNR, we produced the mutant C99A Furthermore,
we reasoned that by eliminating the residue responsible
for enzyme dimerization, we could crystallize PfFNR
as a monomer and thus obtain the three-dimensional
structure of the catalytically active enzyme form The
purified PfFNR-C99A showed the same spectral and
kinetic properties as the wild-type enzyme (not shown)
The titration of the sulfhydryl groups of wild-type and
mutant reductases in the presence and absence of 6 m
guanidinium chloride confirmed that the mutant had
only five cysteines that were titrable under denaturing
conditions and had lost the only –SH group that was
titrable under native conditions in the wild-type
PfFNR As expected, no dimer formation was ever
seen, even after incubation with NADP+and diamide,
a strong oxidant of sulfhydryl groups Unfortunately,
notwithstanding several trials, the mutant proteins did
not yield crystals
PfFNR-bound FAD reduction in anaerobiosis
The enzyme solution was made anaerobic and, after
recording of the spectrum of the oxidized species, a
1.2 molar excess of NADPH was added from the
side arm of the anaerobiosis cuvette Immediately,
the yellow solution was bleached and a
long-wave-length band appeared, extending over 800 nm
(Fig 3A) This absorbance band can be tentatively assigned to a charge transfer complex between fully reduced enzyme-bound FAD and NADP+ (CT2), on the basis of previously described reduced species of other FNRs [19–21] No FAD semiquinone was observed, even during the slow reoxidation of the enzyme by oxygen that occurred after the cuvette had been opened to the air
Stepwise anaerobic photoreductions of the FAD prosthetic group of PfFNR were performed in the presence and absence of NADP+ Again, at variance with other FNRs, unliganded PfFNR was reduced to the hydroquinone level without any transient accumu-lation of FAD semiquinone (Fig 3B) In the presence
of an approximately equimolar amount of NADP+, during reduction the long-wavelength band extending
C
Fig 1 Effect of NADP+on the hydrogen peroxide-promoted dimer-ization of PfFNR PfFNR ( 15 l M ) was incubated at 20 C in the presence of 0.5 m M H2O2in 50 m M Tris ⁄ HCl (pH 7.4) containing 10% glycerol (A, B) The progress of protein dimerization either in the absence (A) or in the presence (B) of 1 m M NADP + , as analyzed
by nonreducing SDS ⁄ PAGE on 10% polyacrylamide gels Incubation times are indicated above the respective gel lanes The molecular masses of the protein standards (M) are: 205, 116, 97.4, 66, 34, and 29 kDa, from top to bottom (C) Time course of the NADPH-ferricyanide reductase activity of PfFNR incubated either in the absence (open circles) or in the presence (filled circles) of 1 m M
NADP + Curves represent the equations of single exponential decays with nonzero endpoint values fitted to the experimental data points.
Trang 5over 800 nm, described above, was transiently seen.
Again, no FAD semiquinone species were observed
during the reaction (Fig 3C) From the absorption
spectra recorded in the latter type of experiment, the
concentrations of the oxidized and reduced species of
the two redox couples (PfFNRox⁄ PfFNRred and
NADP+⁄ NADPH) were measured after each
irradia-tion step Nernst plots yielded Emvalues for the
two-electron reduction of the FAD bound to PfFNR of
)280 ± 2 mV at pH 7 and of )298 ± 4 mV at pH 8
Rapid reaction study on the reductive
half-reaction
Steady-state kinetic analysis of the ferricyanide
reduc-tase activity of PfFNR suggested that the reductive
half-reaction of the PfFNR catalytic cycle was
probably responsible for the low value of kcat[13] To
test this hypothesis and to identify the catalytic step(s) actually involved, the process of reduction of PfFNR-bound FAD by NADPH was studied by stopped-flow diode array spectrophotometry Experimental condi-tions were chosen in order to determine values that
A
B
C
Fig 3 Redox properties of PfFNR-bound FAD Anaerobic solutions
of 15 l M PfFNR in 50 m M Hepes ⁄ NaOH (pH 7.0) containing 10% glycerol were either reacted with a slight excess of NADPH or photoreduced either in the absence or in the presence of a slight excess of NADP + All reactions were carried out in anaerobic cuvettes thermostated at 15 C (A) The spectrum of the PfFNR solution was recorded before (thin line) and after (bold line) the addition of NADPH in a 1.2 : 1 molar ratio to the enzyme from the cuvette side arm (B) The spectrum of the PfFNR solution, con-taining 13 m M EDTA and 1.3 l M 5-deaza-riboflavin, was recorded before (thin line) and after (bold lines with different styles) succes-sive light irradiation periods (C) Spectra were recorded under the same conditions described for (B), with the exception that NADP +
was included in the reaction mixture in a 1.3 : 1 molar ratio with the enzyme The arrows indicate the direction of the spectral changes observed as irradiation time increased In the presence of NADP + , the intensity of the smooth band centered at about 800 nm was maximal when the absorbance of the bands at 380 and 450 nm was almost completely bleached (bold line) Further irradiation led
to the complete disappearance of absorption above 550 nm.
A
B
Fig 2 Effect of dihydrolipoate on the dimeric form of PfFNR Gel
filtration-purified dimeric PfFNR ( 15 l M FAD) was incubated at
20 C in the presence of 50 l M dihydrolipoate in 50 m M Tris ⁄ HCl
(pH 7.4) containing 10% glycerol (A) Analysis of the reaction
mix-ture by nonreducing SDS ⁄ PAGE on a 10% polyacrylamide gel
Incu-bation times are indicated above the respective gel lanes The
molecular masses of the protein standards (M) are: 205, 116, 97.4,
66, 34 and 29 kDa, from top to bottom (B) Time course of the
NADPH-ferricyanide reductase activity of PfFNR The curve
repre-sent a single exponential decay equation fitted to the experimental
data points.
Trang 6were directly comparable to data already published for
plant FNRs As a consequence, the solution medium
of rapid reaction studies differed from that used in
steady-state assays in composition, pH, and ionic
strength PfFNR ( 38 lm before mixing) was reacted
at 25C with NADPH at concentrations ranging from
50 lm to 2 mm (before mixing) Time-resolved spectra
were collected between 300 and 700 nm The spectra
of PfFNR reacting with 25 lm NADPH (after mixing)
are shown in Fig 4A Within the instrument
dead-time, the formation of a detectable amount of charge
transfer complex between NADPH and enzyme-bound
FAD (CT1) occurred, as judged from the high A550 nm
of the spectra recorded immediately after mixing, and
from the value of A450 nm, which was lower than
expected for oxidized PfFNR The observable part of
the reaction consisted of a further bleaching of the
fla-vin main absorbance band (at 450 nm) with a
concom-itant decrease in the absorbance band around 550 nm
Another spectral change occurring during this phase
was a slight increase in the absorbance at wavelengths
above 650 nm, indicating the accumulation of a small
amount of CT2 at the end of the reaction (Fig 4A)
The analysis of a series of shots at different NADPH
concentrations showed that the amount of CT1
observed after the dead-time increased with the
con-centration of NADPH, whereas the CT2 present at the
reaction endpoint followed the opposite trend, being
undetectable when the enzyme was reacted with
reduc-tant concentrations higher than 100 lm (Fig 4B) We
concluded that the observable part of the reaction
occurring under NADPH excess corresponded to the
conversion via hydride transfer of CT1 to the complex
between reduced PfFNR and NADPH, with
accumula-tion of the CT2 intermediate only when the
concentra-tion of NADPH was low, according to the following
scheme:
PfFNRoxþNADPH$CT1$CT2$PfFNRredþNADPþ
PfFNRredþNADPH$PfFNRred=NADPH
According to this reaction mechanism, high
NADPH concentrations would displace NADP+from
its complex with PfFNRred(CT2), by favoring the
for-mation of the PfFNRred–NADPH complex
Absor-bance traces at different wavelengths were monophasic
and fitted well to single exponential decay equations,
yielding the same apparent rate constant (kobs) for any
NADPH concentration Global fitting over the entire
wavelength range confirmed that all of the observed
spectral changes were due to a single process A typical
result of the analysis of an absorbance trace is shown
in Fig 4C A plot of kobs as a function of [NADPH] gave an upper limiting value for this constant of
125 s)1(inset of Fig 4C) As shown in the same figure,
kobs was found to be essentially independent of NADPH concentration when it is higher than 50 lm, suggesting a Kd for the PfFNR–NADPH complex in the lower micromolar range
A
B
C
Fig 4 Reductive half-reaction of PfFNR as studied by stopped-flow diode array spectrophotometry PfFNR was reacted anaerobically with NADPH at 25 C in 50 m M Hepes (pH 7.0) The enzyme concentration was 19 l M, and the NADPH concentration ranged from 25 l M to 1 m M (after mixing) (A) Reaction of PfFNR with
25 l M NADPH (after mixing) Spectra recorded 1 ms (red), 5 ms (green), 9 ms (brown), 13 ms (magenta), 21 ms (yellow) and 51 ms (blue) after mixing are shown The spectrum of the oxidized enzyme mixed 1 : 1 with the above buffer is reported for compari-son (black trace) (B) Reaction of PfFNR with 500 l M NADPH (after mixing) The spectra shown were recorded at the same reaction times as the traces shown in (A) The insets of (A) and (B) show enlargements of the portions above 520 nm of some of the spectra shown in the respective panels (C) Time course of the same reac-tion shown in (B) The curve represents the equareac-tion of a single exponential decay fitted to the experimental data points, yielding a
k app of 122 ± 3 s)1 The inset shows the plot of the k app value as a function of the NADPH concentration (after mixing).
Trang 7Interaction between PfFNR and PfFd
We have investigated the protein–protein interactions
through different approaches Carbodiimide-promoted
crosslinking has been extensively used in the past
[22–24] Indeed, it allows the isolation of covalent
complexes of the two proteins under study that can be
further analyzed Both T gondii Fd and PfFd were
crosslinked to PfFNR with
1-ethyl-3-[3-dimethylamino-propyl]carbodiimide hydrochloride (EDC), yielding a
single band of approximately 49.5 kDa in SDS⁄ PAGE
This molecular mass value is consistent with a 1 : 1
stoichiometry of the proteins in the covalent complex
Apparently, the presence of NADP+did not influence
the crosslinking reaction course In contrast to the
spinach FNR–Fd cross-linked complex [22], the
P falciparum covalent complex does not gain the
capacity to transfer electrons from NADPH to
cyto-chrome c These results could be explained by
measur-ing the diaphorase activity of the reductase durmeasur-ing the
crosslinking reaction Whereas PfFNR alone
main-tained more than 90% of its diaphorase activity after
carbodiimide treatment, inactivation ensued when it
was incubated with the crosslinker in the presence of
PfFd The inactivation was not due to protein–protein
crosslinking, because full inactivation was reached with
only 30–40% covalent complex produced It is possible
that EDC can modify a reductase essential residue(s)
only when PfFNR forms a complex with PfFd
It is well known that ligands of plant-type FNRs
induce perturbations in their visible absorption spectra
The difference absorption spectra obtained by titrating
PfFNR with PfFd allowed determination of the Kd of
the protein complex A positive difference spectrum
was observed, as in the case of spinach FNR, with the
same De of 2.8 mm)1Æcm)1 at 460 nm [25]; however,
the peak in the 300–400 nm wavelength range was less
intense and defined Several titrations were performed
at pH 7, with the ionic strength of the medium being
varied to allow extrapolation of the complex Kdvalue
at 0 ionic strength (Fig 5) This value falls in the
nanomolar range At a more physiological values of
ionic strength of 50–100 mm, the Kd values range
between 0.1 and 0.5 lm, which indicates high affinity
between the two oxidized proteins
We found that PfFd prevents the formation of the
covalent dimer of PfFNR This was shown by
moni-toring the inactivation of the diaphorase activity of
the enzyme incubated in the presence of both
NADP+ and PfFd for more than 1 day The
amount of dimer formed was negligible in
compari-son to that formed in the absence of PfFd (data not
shown)
Discussion
The catalytic properties of PfFNR will be discussed here using the well-characterized FNRs from both plants and T gondii as reference, in order to highlight the peculiar properties of the malarial parasite enzyme The catalytic efficiencies of PfFNR, in both the diaph-orase and Fd–reductase reactions, were found to be significantly higher at pH 7 than at pH 8.2, the latter being the pH optimum for plant and T gondii FNRs The improvement of this kinetic parameter at lower
pH is due to a substantial decrease in the Km values for both NADPH and Fd Better binding of NADPH
at pH 7 can be ascribed to the protonation of His286,
Fig 5 Effect of the ionic strength on the interaction between PfFNR and PfFd PfFNR titrations with PfFd were carried out at
16 C in 50 m M Hepes ⁄ NaOH (pH 7.0) containing different concen-trations of NaCl Complex formation was quantified spectrophoto-metrically on the basis of the perturbations of the absorption spectra of prosthetic groups induced by PfFd binding to PfFNR Upper panel: titration curves obtained in the presence of 50 m M
(filled circles), 100 m M (open circles), 150 m M (filled squares),
200 m M (open squares) and 300 m M (filled triangles) NaCl Curves represent the theoretical equations for 1 : 1 binding fitted to each experimental dataset by optimizing the Kdvalues Lower panel: plot
of the calculated Kdvalues of the PfFNR–PfFd complex as a func-tion of the ionic strength of the medium.
Trang 8which was shown to make contact with the
5¢-phos-phate of the 2¢-P-AMP moiety of the nucleotide
substrate (Fig 6A) [13] This His is a peculiar,
Plasmo-dium-specific replacement for the aliphatic residue
(usually Leu) present at this position in plastidic-type
FNRs [26,27], including the T gondii enzyme [5] It is
important to recall that the NADP-binding site of
PfFNR differs substantially from that of typical
plasti-dic-type FNRs Indeed, PfFNR lacks the two highly
conserved positive side chains that bind the
2¢-phos-phate moiety of NADP (Fig 6A) [6,13] PfFNR is also
distinguished from other plastidic-type FNRs by the
kcat values for both the diaphorase and the
Fd–reduc-tase reactions Such differences cannot arise from a
wrong choice of the first residue of the mature PfFNR
polypepdide, because we have previously shown that
the diaphorase activities of both spinach and T gondii
enzymes were not affected by the length of their
N-ter-minus [9,28] Furthermore, a longer PfFNR (starting
at Leu38 of the precursor) was found to be even less
catalytically active [14] In all FNRs, the rate of the
ferricyanide reductase reaction was shown to be
lim-ited by hydride transfer from NADPH to FAD For
this reason, we investigated this process in PfFNR by
rapid kinetics As expected, the kobs at 25C for
hydride transfer from NADPH to FAD (250
e)eqÆs)1) was comparable to the kcatof PfFNR in the
NADPH–ferricyanide reaction The impairment of the
hydride transfer rate in PfFNR could tentatively be
ascribed to the substantial alteration of the canonical
NADP(H)-binding site of the malarial parasite FNR
It is possible that the peculiar interaction of the
PfFNR with the adenylate moiety of NADP(H) could either slow down the entry of the nicotinamide ring into the active site or alter its positioning with respect
to the isoalloxazine ring of FAD A similar hypothesis has recently been proposed to explain the very low hydride transfer rate of the bacterial-type FNR of Rhodobacter capsulatus [21]
No simple explanation seems to hold in the case of the low rate of steady-state PfFd reduction, where the
kcatof PfFNR is five-fold lower than those of plant and
T gondii FNRs In PfFNR, the bound FAD possesses
an Emsuited for electron flow towards the [2Fe–2S] clus-ter of PfFd [14] Kmand Kdvalues for PfFd are similar
to those reported for other plastidic-type FNR⁄ Fd cou-ples [10,29,30] On the other hand, FAD semiquinone is highly destabilized in PfFNR It is thus possible that one-electron transfer to Fd is impaired because of the high energy required to generate the flavin semiquinone Further studies are required to clarify this point
We have further analyzed the specific property of PfFNR, unparalleled in other FNRs, to undergo dimer-ization in the presence of NADP+or its analogs, with concomitant inactivation We have already proposed a mechanism for this process, which provide a rationale both for the lack of catalytic activity of the dimeric protein and for the role of NADP+ or 2¢-P-AMP in favoring the formation of the intersubunit disulfide bridge [6,13] Briefly, binding of NADP+or its analogs
to monomeric PfFNR promotes an induced-fit transi-tion consisting of the partial unwinding of the aF helix (Fig 6A) and other minor conformational changes In the ligand-bound form of PfFNR, the formation of a
Fig 6 Crystal structure of the PfFNR dimer and details of the interaction between the enzyme and 2¢-P-AMP (A) Ribbon model of an enzyme subunit of the 2¢-P-AMP-bound dimeric form of PfFNR (Protein Data Bank accession no 2OK7, chain A) [13] FAD, 2¢-P-AMP, Cys99, which is involved in the covalent stabilization of the dimer, and the residues most directly involved in the binding of the ligand are shown as wire frames The aF helix, which undergoes a large conformational transition upon ligand binding, is indicated The FAD-binding and the NADP-binding domains of the protein are shown in yellow and blue, respectively (B) Ribbon model of the dimeric form of PfFNR (Protein Data Bank accession no 2OK7, chains A and B) [13] The two subunits are shown in different colors The two bound molecules of the substrate analog 2¢-P-AMP and the disulfide bridge linking the Cys99 residues of the two subunits are indicated The two FAD prosthetic groups can be seen as wire frame models in the central portion of the dimeric assembly.
Trang 9homodimer, in which the Cys99 residues from the two
interacting subunits are placed close together, is highly
favored In the presence of O2, such thiols are
con-verted to a disulfide that locks the dimer (Fig 6B) We
have previously reported that the disulfide-linked
dimeric PfFNR is inactive as a diaphorase, because it
adopts a conformation in which the active site is poorly
accessible to the solvent (Fig 6B) [13] Here, we
showed that dimerization fully abolished the
Fd-depen-dent physiological activity of PfFNR Besides oxygen,
other physiological oxidants have been tested for their
ability to promote dimerization Both hydrogen
perox-ide and oxidized lipoic acid were able to yield high
amounts of PfFNR dimer in relatively short times,
especially in the presence of NADP+ Conversely,
di-hydrolipoic acid and, although less efficiently, GSH
could convert the inactive dimer back to the active
monomer Whether this dimerization⁄ inactivation is
operating in the Plasmodium apicoplast remains to be
checked However, the fact that oxidants and
reduc-tants that are possibly present in the organelle actually
promote in vitro monomer–dimer interconversion
sug-gests that this process could be relevant in vivo
We have produced the C99A variant of PfFNR and
shown that it lacks the single titrable Cys observed in
the wild-type enzyme under native conditions, thus
identifying Cys99 as this residue Furthermore,
PfFNR-C99A possesses the same functional properties
as wild-type PfFNR, with exception of the ability to
undergo dimerization, which is completely abolished in
the mutant PfFNR-C99A is expected to represent a
useful model of the wild-type enzyme in studies where
the possible complications due to dimerization⁄
inacti-vation of PfFNR need to be avoided In particular, it
will be a valuable tool in the screening of chemical
libraries to search for PfFNR inhibitors, allowing
dis-crimination between compounds that target its active
site and compounds that cause its dimerization
Experimental procedures
NADP+and NADPH were purchased from Sigma Horse
heart cytochrome c (Sigma-Aldrich, Milano, Italy) was
fur-ther purified by cation exchange chromatography on SP
Sepharose (GE Healthcare, Milano Italy) Restriction
endo-nucleases were obtained from GE Healthcare, and factor
Xa from Pierce Biotechnology, Inc (Rockford, IL, USA)
All other chemicals were of the highest grade
Plasmid construction
The cloning of the coding sequence of PfFNR (gene
PFF1115w, located on chromosome 6) and the construction
of the expression plasmid pET-PfFNR have already been reported [7] In brief, the DNA encoding the putative mature PfFNR (starting from Lys56 of the protein precur-sor) was originally cloned from the genomic DNA of P fal-ciparum and inserted in the expression vector pET28b (Novagen, Merck KGaA, Darmstadt, Germany) in order
to be expressed in E coli as a cleavable fusion protein with
an N-terminal poly-His-tag Mutation of Cys99 to Ala was achieved by PCR using the QuikChange II site-directed mutagenesis kit (Stratagene, Agilent Technologies, Cernu-sco sul Naviglio, Milano, Italy) and the oligonucleotides 5¢-CATATTAAAAAACAACGAGCTGCCAGATTATATT CTATATCC-3¢ (sense primer) and 5¢-GGATATAGAATA TAATCTGGCAGCTCGTTGTTTTTTAATATG-3¢ (anti-sense primer), which harbor the desired codon change (underlined bases) The DNA sequence encoding mature PfFd was amplified by PCR from pTUK–PfFd [5], using the oligonucleotides 5¢-CCATGCCATGGCTTTATTTTA TAATATAACATTAAGAAC-3¢ (sense primer) and 5¢-CCGGAATTCTTAATTCATTACATGTCGTG-3¢ (anti-sense primer), which introduced NcoI and EcoRI restriction sites (underlined bases) at the 5¢-end and the 3¢-end of the coding sequence, respectively The fragment was cloned between the same sites of pET28b (Novagen), yielding the plasmid pET–PfFd, which allows the synthesis of mature PfFd with no extra residues The insert of all expression plasmids was entirely sequenced to exclude the presence of artefacts
Overexpression and protein purification For overproduction of PfFNR and PfFNR-C99A, Roset-ta(DE3) (Novagen) E coli cells harboring the plasmids pET–PfFNR and pET–PfFNR-C99A, respectively, were grown at 20C in 2· YT medium supplemented with kana-mycin and chloramphenicol (30 mgÆL)1 each) and induced with 0.1 mm IPTG for 24 h The E coli cell paste was resuspended in buffer A (50 mm NaH2PO4, pH 8, 500 mm NaCl, 5 mm imidazole, 1 mm phenylmethanesulfonyl fluo-ride, 1 mm b-mercaptoethanol) at a ratio of 3 mL of solvent per gram of cell (fresh weight) After cell disruption
by sonication, the clarified cell-free extract was loaded on
an Ni2+–Sepharose high performance column (GE Health-care) pre-equilibrated in buffer A Extensive washing was performed with buffer A containing 20 mm imidazole and 10% glycerol The enzyme was eluted with a 20–500 mm imidazole gradient under the conditions recommended by the resin manufacturer The best fractions were pooled together, 5 mm EDTA was added, and ammonium sulfate was added to 15% saturation, for chromatography on a phenyl–Sepharose high performance column (GE Health-care) Ammonium sulfate in a decreasing gradient of satu-ration from 15% to 0% in buffer B (50 mm Tris⁄ HCl, pH 7.4, containing 10% glycerol and 1 mm dithiothreitol) was
Trang 10used to elute the enzyme The best fractions were pooled
and precipitated at 50% ammonium sulfate saturation The
enzyme was solubilized in buffer B and desalted on a
HiTrap column (GE Healthcare) After incubation for 48 h
at 16C with 1 : 500 (w ⁄ w) factor Xa to cleave off the
His-tag, the mixture was loaded again on the Ni2+–Sepharose
column Owing to the moderate intrinsic affinity of PfFNR
for the nickel ion, the enzyme was retained by the column
even in the absence of the His-tag, and was specifically
eluted with 20 mm imidazole The FNR-containing
frac-tions were concentrated by ultrafiltration, desalted by gel
filtration on a PD10 column (GE Healthcare), and stored
at )80 C in 50 mm Tris ⁄ HCl (pH 7.4) containing 10%
glycerol and 1 mm dithiothreitol For Fd overproduction,
E coli strain Rosetta(DE3) was cotransformed with the
pET–PfFd plasmid and the pRKISC plasmid The latter
plasmid (kindly provided by Y Takahashi, Department of
Biological Sciences, Graduate School of Science, Osaka
University, Japan) harbors the E coli isc gene cluster [31],
which we found to improve the yield of holo-PfFd The
transformed cells were grown at 37C in Terrific Broth
medium supplemented with 30 mgÆL)1 kanamycin,
30 mgÆL)1 chloramphenicol, and 10 mgÆL)1 tetracycline
Induction was achieved with 0.1 mm IPTG for 3 h PfFd
was purified as previously described for recombinant
spin-ach leaf FdI, omitting the final Sephadex G-75 gel filtration
[16] Purified PfFd was stored at )80 C in 150 mm
Tris⁄ HCl (pH 7.4), under nitrogen
Spectral analyses and steady-state kinetics
Absorption spectra were recorded either on an 8453 diode
array (Agilent) or a Cary 100 double-beam (Varian, Leinı`,
Torino, Italy) spectrophotometer The extinction coefficient
of the protein-bound flavin of PfFNR was determined
spec-trophotometrically by quantitating the FAD released from
the apoprotein following SDS treatment [32] Steady-state
kinetic parameters were determined for the K3Fe(CN)6
reductase, NADPH-INT reductase and the cytochrome c
reductase (Fd-dependent) activities as previously described
[9,33] The concentrations of both the electron donor and
the electron acceptor were independently varied within the
following ranges: NADPH, 20–500 lm; K3Fe(CN)6, 0.04–
1 mm; INT, 20–300 lm; and PfFd, 0.5–20 lm Initial rate
data were fitted to the equation for a ping-pong Bi-Bi
mechanism by nonlinear regression using the software
grafit5.0 (Erithacus Software Ltd, Horley, UK)
Analysis of the interconversion between
monomeric and dimeric PfFNR forms
To study PfFNR dimer formation, 15 lm monomeric
enzyme was diluted in 50 mm Tris⁄ HCl (pH 7.4) containing
10% glycerol and incubated at 20C The dimerization
reaction was started by adding either 0.5 mm H2O2or dia-mide as oxidant When NADP+ was present, the concen-tration was 1 mm To study restoration of the monomeric form by disulfide reductants, 7.5 lm gel filtration-purified PfFNR (see below) was incubated with either 50 lm dihy-drolipoate or 100 lm GSH under the same conditions described above Aliquots of the reaction mixtures were withdrawn and analyzed by both enzyme assay and SDS⁄ PAGE Before electrophoresis, 5 mm N-ethylmalei-mide and 4% SDS were added to the samples After
30 min of incubation at room temperature, samples were loaded on 12% polyacrylamide gels, omitting the addition
of b-mercaptoethanol In order to obtain dimeric PfFNR in purified form for further characterization, the enzyme was incubated for 36 h in the presence of NADP+ under the conditions described above, without the addition of oxi-dants The PfFNR dimer formed through Cys99 oxidation
by atmospheric O2was isolated by gel filtration on a Super-dex 75 10⁄ 30 column (GE Healthcare) in 20 mm Tris ⁄ HCl (pH 7.4) containing 100 mm NaCl
Active site titrations and FAD photoreductions Titrations of the PfFNR with PfFd were performed spec-trophotometrically at 15C using a Cary 100 (Varian) double-beam spectrophotometer The enzyme was gel filtered in 20 mm Hepes⁄ NaOH (pH 7.0) containing 10% glycerol, and diluted in the same buffer to a final concentra-tion of approximately 15 lm Mixtures included also NaCl
to vary the ionic strength of the medium Difference spectra were computed by subtracting from each spectrum that obtained in the absence of ligand, corrected to account for dilution Kdvalues were obtained by fitting datasets by non-linear regression to the theoretical equation for 1 : 1 binding [34] Stepwise reduction of the PfFNR was performed by the light–EDTA system [35] in anaerobic cuvettes at 15C The enzyme was diluted to 15 lm in 50 mm Hepes⁄ NaOH (pH 7.0) containing 10% glycerol, 13 mm EDTA, and 1.3 lm 5-carba-5-deazariboflavin The solution was made anaerobic by successive cycles of equilibration with O2-free nitrogen and evacuation When present, NADP+was in a 1.2 molar ratio with respect to the enzyme The latter type
of photoreduction experiment allowed the estimation of the redox potential of PfFNR-bound FAD according to the procedure previously described [36], using the NADP+⁄ NADPH couple as the redox indicator
Rapid kinetics Rapid reaction studies of the enzyme reductive half-reaction were performed under anaerobic conditions using a Hi-Tech Scientific SF-6I DX2 stopped-flow spectrophotometer, equipped with a diode array detector (300–700 nm) Enzyme ( 38 lm, before mixing) was reacted with various