Báo cáo khoa học: Leishmania donovani phosphofructokinase Gene characterization, biochemical properties and structure-modelling studies doc

Leishmania donovani
phosphofructokinase
Gene characterization, biochemical properties and structure-modelling studies
Claudia Lo
´
pez
1,2
, Nathalie Chevalier
2
,Ve
´
ronique Hannaert
2
, Daniel J. Rigden
3
, Paul A. M. Michels
2
and Jose Luis Ramirez
1,4
1
Instituto de Biologı
´
a Experimental, Universidad Central de Venezuela, Caracas, Venezuela;
2
Research Unit for Tropical Diseases,
Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite
´
Catholique de Louvain, Brussels,
Belgium;
3
CENARGEN/EMBRAPA, Brası

´
lia, Brazil;
4
Instituto de Estudios Avanzados-Ministerio de Ciencia y Tecnologı
´
a,
Caracas, Venezuela
The characterization of the gene encoding Leishmania
donovani phosphofructokinase (PFK) and the biochemical
properties of the expressed enzyme are reported. L. donovani
has a single PFK gene copy per haploid genome that encodes
a polypeptide with a deduced molecular mass of 53 988 and
a pI of 9.26. The predicted amino acid sequence contains a
C-terminal tripeptide that conforms to an established signal
for glycosome targeting. L. donovani PFK showed most
sequence similarity to inorganic pyrophosphate (PP
i
)-
dependent PFKs, despite being ATP-dependent. It thereby
resembles PFKs from other Kinetoplastida such as
Trypanosoma brucei, Trypanoplasma borreli (characterized
in this study), and a PFK found in Entamoeba histolytica.It
exhibited hyperbolic kinetics with respect to ATP whereas
the binding of the other substrate, fructose 6-phosphate,
showed slight positive cooperativity. PP
i
,evenathighcon-
centrations, did not have any effect. AMP acted as an acti-
vator of PFK, shifting its kinetics for fructose 6-phosphate
from slightly sigmoid to hyperbolic, and increasing consid-

erably the affinity for this substrate, whereas GDP did not
have any effect. Modelling studies and site-directed muta-
genesis were employed to shed light on the structural basis
for the AMP effector specificity and on ATP/PP
i
specificity
among PFKs.
Keywords: phosphofructokinase; Kinetoplastida; allosteric
regulation; mutagenesis; structure modelling.
Glycolysis is a central metabolic pathway in all organisms.
A key enzyme of this pathway is 6-phospho-1-fructokinase
(PFK) or ATP:
D
-fructose-6-phosphate 1-phosphotransfer-
ase. The activity of this enzyme is, in almost all organisms,
regulated by multiple mechanisms. Whereas most glycolytic
enzymes have been remarkably conserved during evolution,
considerable sequence variability is found among PFKs of
different taxonomic groups [1].
In Kinetoplastida, a taxonomic order of protozoan
organisms that includes important pathogens (species of
Trypanosoma, Leishmania, Phytomonas)toman,animals
and plants, the first seven enzymes of the glycolytic pathway
are confined to an organelle called the glycosome [2,3]. PFKs
can be divided into ATP-dependent and PP
i
-dependent
enzymes; the former use ATP as phospho donor in areaction
that is essentially irreversible under physiological conditions,
whereas the latter use PP

i
in a reversible reaction that can be
near equilibrium in vivo [4]. We have previously reported that
Trypanosoma brucei PFK, despite being an ATP-dependent
enzyme, has an amino-acid sequence typical of PP
i
-PFKs [5].
Furthermore, the activity of the T. brucei enzyme appears
only regulated to a limited extent: effectors that modulate
PFK activity in other organisms (vertebrates, yeast) such as
ATP, citrate, fructose 2,6-bisphosphate, ADP and P
i
, have
no effect on the Trypanosoma enzyme [6,7]. Only activation
by AMP was observed.
We hypothesized that an ancestor of the trypanosomes
must have possessed a PP
i
-dependent PFK that changed its
specificity for phospho donor from PP
i
to ATP during
evolution [5]. The many structural differences between the
active site of the two classes of PFKs, and the striking
differences in ligand-binding properties between the human
and parasite enzymes suggest great potential for structure-
based design of drugs [5,8].
For comparative purposes we decided to study the
PFK of Leishmania donovani another kinetoplastid
organism that, contrary to the bloodstream-dwelling

T.brucei, lives as an intracellular parasite in humans.
The results of this work are presented in this paper,
together with a report of the cloning and analysis of the
PFK gene of the fish parasite Trypanoplasma borreli (a
representative of the Bodonina, a different sub-order of
the Kinetoplastida).
Correspondence to J. R. Ramirez, Instituto de Biologı
´
aExperimental–
UCV, Calle Suapure, Colinas de Bello Monte, Caracas, Venezuela,
Caracas 1041-A, Venezuela.
Fax: + 58 221 962 1120, Tel.: + 58 221 751 0111,
E-mail:
Abbreviations: PFK, 6-phosphofructokinase; ATP-
PFK, ATP-dependent phosphofructokinase; PP
i
-PFK, PP
i
-dependent
phosphofructokinase; PTS, peroxisome-targeting signal.
Enzymes: 6-phosphofructokinase (EC 2.7.1.11); pyrophosphate–
fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90).
Note: The novel nucleotide sequences reported in this paper have been
deposited in the EMBL, GenBank and DDBJ databases and are
available under the accession numbers AY029213 (L. donovani PFK)
and AJ310928 (T. borreli PFK).
(Received 11 June 2002, accepted 1 July 2002)
Eur. J. Biochem. 269, 3978–3989 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03086.x
MATERIALS AND METHODS
Cloning and characterization of the

L. donovani
and
T. borreli
PFK genes
A genomic library of L. donovani (kindly donated by
T. deVos, Seattle Biomedical Research Institute, WA,
USA) constructed in Supercos7 vector was screened with
a 500 bp fragment of the PFK gene of T. brucei as a probe
[5]. One positive colony was chosen and the bacteria were
grown for purification of the recombinant cosmid DNA.
Southern analysis using the T. brucei probe showed
hybridization with a 500 bp Sau3AI fragment and a 12 kb
NotI fragment. The Sau3AI fragment was cloned in
pBluescript II KS
–
(Stratagene) and subsequently
sequenced. The DNA sequence obtained had 55% identity
with the corresponding portion of the T.bruceiPFK gene.
This sequence was used as a probe, and to design primers
for further experiments. The NotI fragment was gel purified
anddigestedwithEcoRI, and a 6.5 kb EcoRI fragment
recognized by the Sau3AI probe was subcloned in pBlue-
script II KS
–
(PFK6.5-PBSKS). Finally, from the recom-
binant PFK6.5-PBSKS, a 2.2 kb EcoRI–PvuII fragment
containing the entire L. donovani PFK gene (Fig. 1) was
subcloned in pBluescript II KS
–
(PFK2.2-PBSKS) and

sequenced.
DNA sequences of both strands of recombinant plasmid
inserts were determined by using the T7 DNA polymerase
kit (Amersham Pharmacia Biotech) and [
35
S]dATP (NEN
Life Science Products), or with the Thermo Sequenase
fluorescent labelled primer cycle sequencing kit (Amersham
Pharmacia Biotech), and LI-COR automated DNA
sequence equipment.
A genomic library of T. borreli constructed in kGEM11
(Promega) [9] was screened with a probe consisting of the
entire T. brucei PFK gene [5]. The probe was hybridized
under moderately stringent conditions: 3 · NaCl/Cit/0.1%
SDS, in the presence of 10% dextran sulphate, at 60 °C
(1 · NaCl/Cit ¼ 150 m
M
NaCl/15 m
M
sodium citrate,
pH 7.0). Post-hybridization washes were carried out at
60 °Cfor2hwith5· NaCl/Cit/0.1% SDS, followed for
1h with 3· NaCl/Cit/0.1% SDS. Ten positive recom-
binants were purified and rescreened. High-titre phage
lysates were prepared and DNA was purified from
phages as described previously [10]. From one recombi-
nant phage a 4 kb EcoRI fragment recognized by the
T. brucei probe was subcloned in plasmid pZErO-2
(Invitrogen) and sequenced.
A multiple alignment of the amino-acid sequences of

ATP-PFKs and PP
i
-PFKs was made as described
previously [5].
Structural analysis
MODELLER
[11] was used to construct a model of
L. donovani PFK based on the known structure of
Escherichia coli PFK (PDB code 4pfk [12]). This was
the most suitable template, given our interest in both
catalytic and regulatory sites, among the various available
structures of E. coli and Bacillus stearothermophilus PFK,
as this 4pfk structure contains fructose 6-phosphate and
ADP in the active site, and ADP in the effector site. The
E. coli and L. donovani enzymes share 23% sequence
identity overall, although functional considerations have
led to much greater conservation of the catalytic site and
effector site. Without these sites, the degree of sequence
identity of  20% leads to uncertainty in alignment of
some regions. However, the conservation of the catalytic
and effector sites allows reliable alignment and corres-
pondingly accurate modelling of the Leishmania enzyme in
these regions of particular importance. The program
O
[13] was used for visualizing structures and for its library
of commonly observed, rotameric side-chain conforma-
tions [14].
PROCHECK
[15] was used for stereochemical
analysis of models and for identifying the most likely

position of an important one residue insertion, in the
L. donovani enzyme relative to that from E. coli,atthe
effector site.
Expression and purification of recombinant
L. donovani
PFK
The following specific primers were synthesized to amplify
the gene by PCR: (1) 5¢-CGAATCTC
CATATGGAGA
CTCG-3¢, containing a NdeI site (underlined) adjacent to
the 5¢ end of the coding region; (2) 5¢-
TA
GGATCCTTACACCTTAGACGCCAG-3¢, comple-
mentarytoa3¢ noncoding region followed by a BamHI
site (underlined). The total volume of the amplification
mixture was 100 lL containing 20 ng of DNA, 0.4 l
M
of
each primer, 4 m
M
MgSO
4
, 200 l
M
each of the four
deoxynucleotides, and 1 unit of Vent DNA polymerase with
the corresponding 1 · ThermoPol Reaction Buffer (New
England Biolabs). PCR was performed in a Hybaid
Thermal Reactor (Hybaid, UK) using the following
program: 5 min 95 °C; 30 cycles consisting of 1 min

denaturation at 95 °C, 45 s annealing at 65 °Cand
1.5 min extension at 72 °C; and a final 5 min incubation
at 72 °C. The amplified gene was purified and ligated to the
pCR2.1-TOPO vector (Invitrogen). The resulting recombin-
ant plasmid (PFKLd-TOPO) was used to transform E. coli
strain XL-1 Blue, and the sequence of the insert was
checked.
A bacteriophage T7 RNA polymerase system [16] was
used to express L. donovani PFK in E. coli.ThePFK
gene was excised from the PFKLd-TOPO recombinant
plasmid and spliced into expression vector pET28b using
the NdeIandBamHI sites. The new recombinant
plasmid named pLdPFK directs, under the control of
the T7 promoter, the production of a fusion protein
bearing a N-terminal extension of 20 residues including a
(His)
6
-tag.
E. coli strain BL21(DE3)pLysS transformed with
pLdPFK was grown at 30 °C in 50 mL Luria–Bertani
medium plus 1
M
sorbitol and 2.5 m
M
betaine [17]
Fig. 1. Restriction map of recombinant plasmid PFK6.5-pBSKS. The
hashed box marks the open-reading frame of the L. donovani PFK
gene. The ATG of the initiator methionine is indicated. T7 and T3
indicate the orientation of the insert with respect to the promoter
sequences of the pBSKS vector. The 2.2 kb EcoRI–PvuII fragment

was used as hybridization probe and for sequence analysis.
Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3979
supplemented with 30 lgÆmL
)1
kanamycin and
25 lgÆmL
)1
chloramphenicol. Expression was induced at
an A
600
of 0.6–0.8 by the addition of 1 m
M
isopropyl thio-
b-
D
-galactoside and growth was continued overnight. Cells
were collected by centrifugation (12 000 g,10minat
4 °C). The cell pellet was resuspended in 20 mL of lysis
buffer (50 m
M
triethanolamine/HCl, pH 8.0, 300 m
M
NaCl, 200 m
M
KCl, 1 m
M
KH
2
PO
4

,5m
M
MgCl
2
, 10%
glycerol, 0.1 m
M
fructose 6-phosphate, 0.3 m
M
glucose
6-phosphate and the protease inhibitors E64, leupeptin
and pepstatin, each at a concentration of 1 l
M
). Cells
were lysed by two passages through a SML-Aminco
French pressure cell (SML Instruments, USA) at 90 MPa.
Nucleic acids were eliminated first by incubation with 500
units Benzonase (Merck, Germany) for 15 min at 37 °C,
and then with 10 mg of protamine sulphate for 15 min at
room temperature. The lysate was centrifuged (20 000 g
15 min at 4 °C) and the supernatant used to further purify
the expressed enzyme using immobilized metal affinity
chromatography (TALON resin, Clontech, USA) exploit-
ing the (His)
6
-tag at the N-terminus of the PFK. The
charged resin was washed with lysis buffer plus 10 m
M
imidazole. The enzyme was then eluted with 100 m
M

imidazole in lysis buffer. One millilter fractions were
collected to measure enzyme activity and to determine the
protein profile by SDS/PAGE.
Site-directed mutagenesis of the L. donovani PFK gene
was performed by PCR techniques as described by
Mikaelian & Sergeant [18] and using Vent DNA polymerase.
The Leishmania PFK Lys224 codon AAG was changed into
the Gly codon GGG. The mutated protein was expressed
and purified according to the same protocols as the wild-
type enzyme.
Enzyme assays and kinetic analysis
The activity of PFK was determined by measuring the
decrease of NADH absorbance at 340 nm using a Beckman
DU7 spectrophotometer. To follow PFK during purifica-
tion, a standard enzymatic assay was performed at 25 °Cin
a 1 mL reaction mixture containing: 100 m
M
triethanol-
amine/HCl buffer, pH 8.0, 2.5 m
M
MgSO
4
,10m
M
KCl,
2m
M
fructose 6-phosphate, 0.5 m
M
ATP, 2.2 m

M
PEP,
1.6 m
M
AMP, 0.42 m
M
NADH, 2 U lactate dehydrogenase
and 2 U pyruvate kinase. One activity unit is defined as the
conversion of 1 lmol substrate per min under these
standard conditions.
For kinetic analyses an assay was used in which the
PFK activity was not coupled to a NAD-dependent
reaction through its product ADP, as in the standard
assay, but through its product fructose 1,6-bisphosphate.
The assay was performed at 25 °C in a 1 mL reac-
tion mixture containing 100 m
M
triethanolamine/HCl,
pH 8.0, 2.5 m
M
MgCl
2
,0.42m
M
NADH, 0.4 U aldo-
lase, 0.8 U glycerol-3-phosphate dehydrogenase and
20 U triosephosphate isomerase. The reaction was initi-
ated by the addition of 5 lL of enzyme diluted in buffer
(0.1
M

triethanolamine/HCl, pH 7.4, BSA 0.1 mgÆmL
)1
,
EDTA 0.2 m
M
and dithiothreitol 0.5 m
M
). The effect of
the fructose 6-phosphate concentration was determined
by fixing the ATP concentration at 1 m
M
,inthe
presence and absence of AMP (1.5 m
M
) and GDP
(1.0 m
M
).
RESULTS AND DISCUSSION
Analysis of kinetoplastid PFK genes
In the 30 kb insert of the cosmid obtained by screening a
L. donovani genomic library, only a single gene copy of
PFK was detected. Figure 1 shows a restriction map of the
insert of recombinant plasmid PFK6.5-pBKS subcloned
from that cosmid. The coincidence between the restriction
pattern of this cosmid and that obtained by Southern
analysis of whole Leishmania DNA, and the signal inten-
sities of the bands (not shown), were consistent with the
presence of one gene copy per haploid genome. When blots
of L. donovani chromosomal bands separated by pulsed-

field gel electrophoresis were hybridized with an EcoRI–
PvuII fragment from recombinant PFK6.5-pBSKS (Fig. 1),
a unique hybridization signal of 1.3 Mb was observed
(Fig. 2, lanes 1 and 2). This 1.3 Mb band may correspond
to chromosomes 27b, 28 or 29 [19]. In L. amazonensis,
included for comparative purposes, the probe hybridized
weakly to a band of approximately 1.7 Mb (Fig. 2, lanes 3
and 4), the size of the proposed fusion product of
chromosomes8and29intheL. mexicana group [20].
The amino-acid sequences encoded by the ORFs found
in the L. donovani and T. borreli recombinants are shown
in Fig. 3. The ORF in L. donovani is 1461 bp, coding for a
polypeptide of 486 amino acids (excluding the initiator
methionine) with a molecular mass of 53 988 and a
calculated isoelectric point (pI) of 9.26. The C-terminus has
the tripeptide -SKV, a type 1 peroxisome-targeting signal
(PTS-1) with an acceptable degeneracy of the canonical
motif -SKL [21,22]. The same tripeptide was previously
found in another glycosomal enzyme of this organism,
namely hypoxanthine-guanidine phosphoribosyltransferase
Fig. 2. Chromosomal assignment of the PFK genes in L. donovani and
L. a mazon ens is. Southern blot of Leishmania chromosomal bands
separated by pulsed-field gel electrophoresis after hybridization with a
probe consisting of a radioactively labelled EcoRI–PvuII restriction
fragment (see Fig. 1) containing the whole L. donovani PFK gene.
Lanes 1 and 2, L. donovani, lanes 3 and 4, L. amazonensis.Theposi-
tions of yeast chromosomes that were used as molecular size markers
are indicated at the right-hand side.
3980 C. Lo
´

pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
[23]. The sequence predicted from the T. borreli ORF codes
for a polypeptide of 489 amino acids (excluding the first
methionine) with a molecular mass of 53 211 and a pI 8.9.
The typical PTS-1 motif -SKL was found as the C-terminal
tripeptide. An excess of positively charged residues and
resulting high pI are features often associated with
glycosomal enzymes, particularly in T. brucei [24,25].
Figure 3 also shows the alignment of L. donovani and
T. borreli PFK sequences with those of some other
organisms. Except for two N-terminal insertions in
T. borreli PFK, the new sequences share the characteristics
of T.brucei PFK as described previously [5]. The
percentage identity in a pairwise comparison (Table 1)
of the amino acid sequences of L. donovani and T.brucei
is high (70%), whereas the value obtained by comparing
T. borreli PFK with the L. donovani and T.brucei
enzymes is 54% in both cases. The extent of sequence
similarity among the Kinetoplastida PFKs corresponds
with the values found for some other glycolytic enzymes
[9,26] and with the proposed phylogeny of this order
[27,28]. The percentage identities of the kinetoplastid
PFKs with those from all other major taxonomic groups
are in the range 15–30%. As already previously observed
for the T. brucei PFK [5], the L. donovani and T. borreli
enzymes show signatures typical of PP
i
-PFKs (see Fig. 3)
and, in a phylogenetic analysis, they appear firmly placed
within the cluster of the PP

i
-dependent enzymes, well
separated from the nonkinetoplastid ATP-PFKs (not
shown). Interestingly, the kinetoplastid PFKs showed the
highest percentage identity (37–38%) with the minor
48 kDa PFK from another protist, Entamoeba histolytica.
Despite the higher overall similarity and its phylogenetic
relationship with the subset of PP
i
-PFKs [5,29,30], it was
recently reported that this E. histolytica PFK uses ATP as
phospho donor (in contrast to the very different 60 kDa
PP
i
-dependent PFK of this organism, see Fig. 3) [31]
similar to the observation previously reported for the
T. brucei enzyme [5]. The PFK activity in Leishmania
species [32–34] and in T. borreli (J.VanRoy,F.Opper-
does, N. Chevalier & P. A. M. Michels, unpublished
results) is also known to be ATP-dependent.
Kinetics of
Leishmania
PFK
The L. donovani PFK was expressed in E. coli with an
N-terminal His-tag and purified for kinetic analysis. The
activity of the enzyme was ATP dependent. No activity (less
than 1%) was observed when PP
i
(at concentrations up to
5m

M
) was used as alternative phospho donor. As reported
previously for PFK of T. brucei [7] and other Leishmania
species [32], the activity of the enzyme depends hyperbol-
ically on the concentration of ATP. The kinetic behaviour
of the expressed PFK was determined as a function of
fructose 6-phosphate at fixed, saturated ATP concentration
(1.0 m
M
), and in the presence or absence of AMP and GDP
(Fig. 4). AMP, ADP and GDP are well-known effectors of
bacterial PFKs; in assays, GDP rather than ADP is often
used as effector, because ADP, being a reaction product,
may act as a competitive inhibitor with respect to ATP. In
the absence of AMP and at low fructose 6-phosphate
concentrations (less than approximately 0.2 m
M
)the
enzyme showed slightly cooperative binding of the
substrate, with a Hill coefficient of 1.41. At higher substrate
concentrations, the enzyme displayed hyperbolic kinetics;
the S
0.5
¼ 3.60 ± 0.48 m
M
. In the presence of AMP, the
curve is hyperbolic over the entire range of substrate
concentrations; AMP has a clear stimulatory effect by
increasing the affinity for fructose 6-phosphate till a
K

m
¼ 0.157 ± 0.028 m
M
. In contrast, GDP has no effect
whatsoever on the activity of the enzyme.
Our data thus showed that the expressed L. donovani
PFK binds its substrate fructose 6-phosphate in a cooper-
ative manner, similar to many other PFKs, such as the
enzymes from mammals [35] and bacteria (reviewed in [36]).
This behaviour, and the increased affinity for the substrate
in the presence of AMP, have also been reported for the
enzymes partially purified from cultured L. donovani and
L. braziliensis [32] and for T.bruceiPFK [6,7]. The observed
cooperative substrate binding and allosteric activation by
AMP suggest a multimeric structure for the enzyme. Indeed,
T. brucei PFKwasshowntobetetrameric[25],likethe
ATP-PFKs of most other organisms [1]. Hyperbolic kinetics
have been reported for the Trypanosoma cruzi enzyme, but
the relevance of this finding may be questioned, because the
authors described an enzyme with a 17 kDa subunit mass
[37]. Despite the relatively high sequence identity of
Kinetoplastida PFK and PP
i
-dependent enzymes, AMP
stimulation has only been described for one PP
i
-dependent
PFK, that from Naegleria fowleri [38]. In this case the AMP
effect was attributed to promoting a more active enzyme
aggregate.

Active site of kinetoplastid PFKs
Table 2 presents a summary of the active-site residues of
E. coli PFK involved in the binding of ADP and fructose
6-phosphate as observed in its crystal structure [12], and the
corresponding residues in the PFKs of human, T.brucei,
L. donovani, T. borreli and in the minor 48 kDa enzyme of
E. histolytica. A comparison of the kinetoplastid and
human PFKs shows four differences in the residues involved
in nucleotide binding and three differences in the residues
involved in the binding of fructose 6-phosphate. The same
Fig. 3. Alignment of L. donovani and T. borreli PFK amino acid
sequences with other ATP and PP
i
dependent enzymes. Sequences include
the ATP-dependent enzymes of T.brucei, E. coli, B. stearothermo-
philus, E. histolytica, the N-catalytic domain of the human muscle
enzyme and the catalytic subunit of S. cerevisiae,andthePP
i
-depen-
dent enzymes of E. histolytica, A. methanolica and P. freundenreichii.
Sequences of yeast and human expanding beyond the N- or C-termini
of Kinetoplastida sequences are not shown. The E. coli and L. dono-
vani enzymes are numbered above and below the alignment, respec-
tively.Symbolsare:blackarrows,b strands; black cylinders, ahelices;
open circles, substrate ATP-binding residues; black circles, fructose
6-phosphate binding residues; black triangles, effector-binding resi-
dues. Boxes mark regions sharing identical residues between either the
set of kinetoplastid and E. histolytica ATP-PFKs and the set of typical
ATP-PFKs, or the kinetoplastid and E. histolytica ATP-PFKs and the
setofPP

i
-PFKs. Residues common to all sequences are in bold +
italic font; bold only is used where there is one disagreement among the
entire sequence set. Residue 224 of L. donovani PFK that was studied
by mutagenesis is indicated by a black triangle underneath the align-
ment. The figure was made using
ALSCRIPT
[53].
Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3981
substitutions occur in the 48 kDa ATP-PFK of E. histolytica
(Fig. 3) [39], as well as in one of the PFKs of the prokaryotic
Spirochaetes Treponema pallidum and Borrelia burgdorferi
(data not shown; GenBank accession numbers AE001195
and AE001172). Indeed, the identity of active-site residues
in all these organisms is in agreement with the branching
order in a phylogenetic analysis based on full-length PFK
sequences [5,29,30]. The PFKs of these organisms form a
well-supported monophyletic cluster within the PP
i
-PFK
subset, well separated from the typical ATP-PFKs [5,29,30].
3982 C. Lo
´
pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The identity of the phospho donor of the PFKs from
T. pallidum and B. burgdorferi has not been established
yet.
The E. coli residues involved in fructose 6-phosphate
binding that have been substituted in the kinetoplastid and
related PFKs are Arg162, Arg243 and His249. The

Fig. 3. (Continued.)
Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3983
corresponding residues found in all these PFKs are Gly, Lys
and Tyr, respectively. Whereas Arg162 seems conserved in
all typical ATP-PFKs, a positively charged residue at the
corresponding position seems not essential for substrate
binding in the PP
i
-PFKs and the atypical ATP-PFKs. The
Arg243 of ATP-PFKs is replaced by Lys in all PFKs of the
subset comprising the Kinetoplastida; apparently, a
positively charged residue at this position is essential in all
ATP-PFKs. It is possibly equivalent to Lys315 of the
PP
i
-dependent PFK of Propionibacterium freundenreichii
(E. coli position 241 in Fig. 3), because Xu et al. [40] have
shown that an alteration of this residue by site-directed
mutagenesis causes a 389-fold increase of the K
m
for
fructose 6-phosphate. The substitution of His249 (E. coli
numbering) by Tyr in the Kinetoplastida is also found in
some PP
i
-PFKs such as the enzymes of P. freudenreichii and
E. histolytica (Fig. 3), and is possibly without much effect.
Out of 10 residues involved in ADP binding in E. coli
PFK, four are not conserved in Kinetoplastida (Table 2).
Strikingly, the ATP-dependent E. histolytica PFK and the

putative ATP-PFKs of two Spirochaetes discussed above
have the same residues as the ATP-dependent kinetoplastid
enzymes [39,41]. Therefore, these residues may be important
determinants for the phospho-donor specificity if, in future
research, the isoenzymes of the Spirochaetes turn out to be
ATP-dependent as well.
In addition to the substrate-binding residues in Table 2,
another active-site residue is of particular interest: the
residue corresponding to E. coli Gly124 is a Lys in the
kinetoplastid ATP-PFKs, in the ATP-dependent isoenzyme
of E. histolytica (see Fig. 3) and in the PFKs of the two
Spirochaetes (not shown). A Gly is typical of ATP-PFKs,
Table 1. Percentage identity of the PFK amino-acid sequences given in Fig. 3.
B. stearo.
Human
muscle-N S. cere N T. bruce i L. donovani T. borreli
E. histo.
ATP
E. histo.
PP
i
A. meth.
B. stearothermophilus 54
Human muscle-N 39 42
S. cerevisiae-N 34 37 46
T. brucei 24 30 17 18
L. donovani 23 29 19 18 70
T. borreli 25 25 18 19 54 54
E. histolytica ATP 25 27 19 21 38 37 38
E. histolytica PP

i
21 25 15 12 16 15 16 18
A. methanolica 37 43 32 33 30 29 26 30 21
P. freudenreichii 23 22 18 18 20 20 20 22 17 22
Fig. 4. Kinetics of recombinant L. donovani PFK with respect to fruc-
tose 6-phosphate. Activity was measured at a fixed ATP concentration
of 1.0 m
M
. Symbols are: j, no additions; d,+1.5m
M
AMP;
m,+1.0m
M
GDP. Values of kinetic parameters (see text) were cal-
culated after optimal curve fitting of the experimentally determined
data using the
SIGMAPLOT
program. The values given in the text
are ± SD for the fit.
Table 2. Amino acid residues involved in binding fructose 6-phosphate
and ADP in E. coli PFK, and the corresponding residues in the
organisms L. donovani, T. bruc ei, T. borreli, E. histolytica-ATP,
T. pallidum and B. burgdorferi. Differences are highlighted in bold.
E. coli Other organisms
Fructose 6-phosphate Thr125 Thr
Asp127 Asp
Asp129 Asp
Arg162 Gly
Met169 Met
Gly170 Gly

Arg171 Arg
Glu222 Glu
Arg243 Lys
His249 Tyr
Arg252 Arg
ADP Gly11 Gly
Tyr41 Tyr
Arg72 Arg
Phe73 Gly
Arg77 (gap)
Asp103 Asp
Gly104 Gly
Ser105 Thr
Met107 Arg
Gly109 Gly
3984 C. Lo
´
pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
whereasaLysispresentinallPP
i
-dependent enzymes. Xu
et al. [40] have shown that a change of this lysine into a
methionine in P. freudenreichii PFK caused a 132-fold
increase in the K
m
for PP
i
and a 490-fold decrease in k
cat
,

providing strong indication for a direct involvement of this
Lys residue in PP
i
binding. From a phylogenetic analysis, we
previously concluded that the kinetoplastid PFKs must
have been derived from a PP
i
-dependent ancestral PFK,
which changed its phospho-donor specificity early in the
evolution of the lineage [5]. However, the Lys is no longer
involved in PP
i
binding, so why has it been retained in
the present-day kinetoplastid PFKs which are all ATP
dependent, and in the ATP-dependent isoenzyme of
E. histolytica? Has it obtained a function in ATP binding,
implying that the mode of nucleotide binding is different in
kinetoplastid PFKs from that in other ATP-PFKs? Or has it
been retained for structural reasons? Relevant to these
questions is the observation that the Lys is also present in
the ATP-dependent enzyme from the actinomycete Strep-
tomyces coelicolor that, in a phylogenetic analysis, also
clusters with the PP
i
-dependent PFKs and that must have
had a common ancestor with the PP
i
-dependent PFKs of
other Actinomycetes such as Amycolatopsis methanolica
[42].

We therefore investigated whether substituting the Lys
would have any effect on the kinetoplastid enzyme. To this
end, we replaced it by Gly in L. donovani PFK. The
resulting LdPFK Lys224fiGly mutant did not display any
activity. In contrast, the corresponding LysfiGly mutant of
T. brucei (TbPFK Lys226fiGly) appeared to be active [43].
Strikingly, this T. brucei mutant showed only a slight
decrease in its affinity for ATP, but the mutation had a
major effect on the enzyme’s behaviour with respect to
fructose 6-phosphate. In the absence of AMP, the S
0.5
for
fructose 6-phosphate was  5m
M
compared to 0.59 m
M
in
the wild-type enzyme. However, the higher substrate affinity
can still be induced by the addition of AMP: K
m
for fructose
6-phosphate ¼ 0.82 m
M
compared to 0.15 m
M
in the wild-
type PFK [43]. It seems that the mutation leads to a local
disruption of the active site with accompanying lowering of
fructose 6-phosphate affinity. This is independent of the
allosteric changes in fructose 6-phosphate affinity as AMP is

capable of similar enhancements of fructose 6-phosphate
affinity in both wild-type and mutant enzymes. In the case
of the L. donovani enzyme, a greater degree of disruption
leads to abolition of fructose 6-phosphate binding, either
through local or global effects.
As discussed previously, the comparative analysis of PFK
sequences suggests that only subtle changes may be required
for a change of phospho donor specificity [5]. This notion
was reinforced by the recent publication [44] describing
mutations in the ATP-PFK of E. coli and the PP
i
-PFK of
E. histolytica, similar to those described above for trypan-
osomatid PFKs. The Gly124fiLys substitution in E. coli
effectively eliminated activity with ATP as a substrate, but
no PP
i
-dependent activity was observed. However, the
reverse Lys201fiGly mutation in the PP
i
-dependent, major
PFK of E. histolytica reduced the k
cat
with PP
i
as the
phospho donor by four orders of magnitude, while having
only a limited effect on the apparent PP
i
affinity of the

residual enzyme activity. Importantly, the performance of
the enzyme with ATP as a phospho donor increased about
eightfold (although this is still 10
5
times less than the
performance of the wild-type enzyme with PP
i
) essentially
by an increase in k
cat
.
Understanding of the role of Lys224 in L. donovani PFK,
and corresponding residues in other PFKs, is hampered by
the lack of a crystal structure with a lysine at this position.
Modelling of the Gly to Lys mutation shows that, without
significant local structural changes, the lysine side chain
becomes entirely buried in the protein interior, with no
possibility of electrostatic interaction with an acidic residue,
a situation essentially unknown in protein structure. One
hypothesis is that, in enzymes containing a lysine at this
position, the peptide bond with the preceding proline adopts
a cis configuration [45]. It is suggestive that proline is
entirely conserved at position 123 (E. coli numbering) when
a lysine is present at position 124, while valine is also
tolerated in other PFKs. Modelling of possibilities for a
L. donovani model containing such a cis peptide bond yields
structures in which the lysine side chain is solvent-exposed
such as that illustrated in Fig. 5. In this structure the lysine
side chain is placed at the heart of the catalytic site. A direct
interaction with substrate ATP seems unlikely from the

kinetic results presented here, although it is unfortunate that
structural inferences may only be drawn from a product-
bound PFK structure (Fig. 5). However, a limited descrip-
tion of a PFK-AMPPNP-fructose 6-phosphate substrate
analogue complex [46] (coordinates not deposited) supports
the notion of a close resemblance between substrate- and
product-bound protein structures. Why then, in contrast,
should PP
i
binding be dramatically affected when this lysine
is mutated in E. histolytica and P. freudenreichii PFKs
[40,44]? The explanation may lie in another residue, clearly
implicated in substrate specificity [44]. Position 104 (E. coli
numbering) is always a Gly in ATP-PFKs and an Asp in
PP
i
-PFKs. Structural examination (Fig. 5) shows that the
presence of any non-Gly residue leads to steric clashes with
the bound nucleotide in its crystallographically observed
Fig. 5. Positions of phospho-donor specificity-determining residues rel-
ative to the catalytic site of E. coli PFK bound to products. Numbering is
according to the E. coli enzyme. The figure was produced using
MOLSCRIPT
[54].
Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3985
position. In the structure of a PP
i
-dependent PFK bound to
substrates, it would be reasonable to expect that the PP
i

substrate binds in the corresponding position as the b-and
c-phospho groups of bound ATP in ATP-dependent
enzymes. However, analysis shows that any rotameric
conformation of an Asp104 side chain leads to positioning
of its negative charge near to the phospho group occupying
the Ôa positionÕ, also negatively charged. Minimum oxygen-
oxygen interatomic distances range from 1.1 to 3.9 A
˚
,
depending on Asp104 rotamer. This electrostatic repulsion
may therefore force the PP
i
into a slightly different
conformation, further from Asp104 and hence nearer to
Lys124. This hypothesis allows an explanation of the
apparent involvement of this lysine in PP
i
binding [40,44]
but not in ATP binding. A more prosaic explanation may
underlie the almost complete lack of PP
i
- or ATP-dependent
PFK activity seen for the Gly124fiLys E. coli mutant [44].
Without a preceding cis peptide bond only side chain
conformations that unfavourably bury the positive charge
of the new Lys are attainable and the protein would
therefore be destabilized. The lack of confirmation of native
fold for the mutant, by CD experiments for example,
suggests that the mutant may have undergone gross
structural changes resulting in loss of activity. The modelled

lysine in the L. donovani modeliswellpackedandnot
apparently well positioned to interact directly with fructose
6-phosphate. These considerations support the previously
advanced explanation of the effects of the Lys224fiGly
mutation in terms of destabilizing local structural changes.
Effector-binding site of trypanosomid PFKs
Table 3 presents a comparison of the residues in B. stearo-
thermophilus and E. coli PFKs involved in the binding of the
allosteric activator ADP with the corresponding residues in
the L. donovani and T.bruceienzymes (according to the
alignment in Fig. 3). A structural comparison of the
residues binding the activator ADP in B. stearothermophilus
PFK and a putative AMP binding mode for the L. donovani,
suggested by modelling, is shown in Fig. 6. This comparison
suggests that the kinetoplastid enzymes may employ the
same region for binding their allosteric activator AMP. The
b-phospho group binding residues of the bacterial enzymes
show the most changes, with the most striking substitution
being the replacement of the Mg
2
+-ligating Glu187 with
Asn. The loss of Mg
2+
and the replacements of Arg25 and
Arg154, both of which electrostatically interact with the
b-phospho group of ADP (Fig. 6A), effectively eliminate
the b-phospho-binding pocket in the trypanosomatid
enzymes. The residues at positions 211 and 213 of the
B. stearothermophilus enzyme that bind the a-phospho
Fig. 6. Comparison of (A) the crystallographically observed effector site of E. coli PFK with bound ADP and (B) the modelled structure of L. donovani

PFK effector site bound to AMP. Ligand and protein are shown in ball-and-stick representation with the exception of the protein backbone, in the
vicinity of the one residue insertion, which is drawn as a tube. Possible hydrogen bonds are shown with dotted lines. The figure was produced using
MOLSCRIPT
[54].
Table 3. Amino acid residues involved in binding the allosteric activator
ADP in B. stearothermophilus and E. coli PFK, and corresponding
residues in T. brucei and L. donovani PFKs. Differences are highlighted
in bold.
B. stearothermophilus E.coli T. brucei L. donovani
Arg21 Arg Arg Arg
Arg25 Arg Leu Leu
Val54 Arg Arg Arg
Gly58 Ser Thr Arg
Arg154 Arg Tyr Tyr
Glu187 Glu Asn Asn
Arg211 Lys His Gln
Lys213 Lys Arg Arg
3986 C. Lo
´
pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
group of ADP (Fig. 6A) are better conserved and may be
involved in binding the phospho group of AMP (Fig. 6B).
The modelling reveals that, in addition to the residue
differences identified by sequence comparisons, other
significant structural differences may exist. The L. donovani
enzyme, along with others from kinetoplastids and the
E. histolytica ATP-dependent PFK, has a one residue
insertion, relative to bacterial enzymes, at around position
287 (L. donovani enzyme numbering). Two possible posi-
tions for this insertion were analysed and one, as shown in

Fig. 6B, found to be favoured for avoiding the positioning
of model residues in unusual areas of the Ramachandran
plot. The altered main chain conformation in the vicinity
causes the Gln287 side chain to intrude into the area
corresponding to the b-phospho-binding site of the bacterial
enzymes. Furthermore, it may form a hydrogen bond with
the phospho group of effector AMP (Fig. 6B). Another
interesting structural difference revealed by modelling
relates to the side chain position of Arg157 in the
L. donovani enzyme which replaces a Ser or Gly in the
bacterial enzymes. Adopting a rotameric conformation, this
residue may fill the space caused by the replacements of
B. stearothermophilus Arg211 with Gln and Arg25 with
Leu. In this position it may hydrogen bond to the phospho
of effector AMP in the L. donovani model structure and
contribute to the positive electrostatic potential of the
effector binding pocket.
CONCLUSIONS
The PFK genes of L. donovani and T. borreli have been
cloned and sequenced. The encoded enzymes show most
similarity to the subset of PP
i
-PFKs, as did the previously
analysed T.brucei PFK. Nevertheless, ATP is the phos-
pho substrate of all these kinetoplastid PFKs. It is
possible that a common ancestral organism changed its
phospho donor specificity during evolution. The currently
available data do not allow us to draw any conclusion as
to how and why the Kinetoplastida and other protists
such as Entamoeba obtained their ATP-dependent, PP

i
-
like PFKs. Did they evolve from a PP
i
-PFK in both
lineages independently, or did they originate in a common
ancestor of these protists? Were they acquired from
Spirochaetes by lateral gene transfer? In this respect, it
may be relevant that phylogenetic studies based on
sequences of other glycolytic enzymes, glyceraldehyde-3-
phosphate dehydrogenase and enolase, showed grouping
of Kinetoplastida (and/or the related Euglenoida) and
Spirochaetes [47–49].
Strikingly, all kinetoplastid PFKs, as well as the
Entamoeba PFK contain a Lys on position 124 (E. coli
numbering), whereas all other ATP-PFKs contain a Gly.
Previous mutagenesis studies have provided strong evidence
that this Lys residue is involved in PP
i
binding. Structure
modelling suggests that the Lys may have been retained in
the kinetoplastid PFKs to maintain the stability of the
active-site structure. These results are supported by muta-
genesis studies. No active L. donovani Lys224fiGly mutant
could be obtained, whereas the kinetic properties of a
corresponding Lys226fiGly mutant of T. brucei PFK
could be interpreted in terms of a destabilized active site.
The L. donovani PFK shows slightly cooperative binding
of fructose 6-phosphate at low concentrations of this
substrate. The enzyme was allosterically activated by

AMP by a significant increase in the affinity for the
substrate. However, trypanosomatid PFKs are not activa-
ted by ADP, in contrast to their counterparts in the bacteria
E. coli and B. stearothermophilus. Modelling studies have
provided a possible structural basis for the AMP specificity.
We have provided evidence for significant structural
differences between trypanosomatid PFK and other ATP-
PFKs including the human enzyme. Such differences were
found in both the active site and the region of the enzyme
presumably involved in effector binding. Indeed, the
differences in the effector-binding site tally with the
apparently low level of activity regulation of trypanosoma-
tid PFK as compared to that of the human enzyme. This
limited regulation of trypanosomatid PFK seems physio-
logically relevant in view of the intraglycosomal localization
of the enzyme and the low permeability of the organelle’s
membrane for many metabolic intermediates that in other
cells act as PFK effectors [3,50]. The structural differences
observed offer great potential for the design or selection of
drugs. Although our computer analysis using a kinetic
model of glycolysis suggested that PFK in bloodstream-
form T. brucei is present in excess [51], we have argued
elsewhere [8,52] that this does not necessarily exclude the
enzyme as a target for selective inhibitors that bind with
high affinity, particularly irreversibly binding inhibitors.
The most important aspects to consider in drug target
selection are that an enzyme should have an essential (or at
least very important) metabolic role and that its structure
should be sufficiently different from that of the correspond-
ing host enzyme. Moreover, metabolism in bloodstream-

form T. brucei is highly specialized, and in many respects
not representative for the infective stages of other trypan-
osomatid parasites such as the trypomastigotes and
amastigotes of Leishmania species and T.cruzi [3].
Therefore, we consider the trypanosomatid PFK as a highly
promising drug target.
ACKNOWLEDGEMENTS
This research was financially supported by the European Commission
(programmes STD3 and INCO-DC). Financial support for C. L. for a
1 year stay at the ICP in Brussels was provided by the Fundacio
´
nGran
Mariscal de Ayacucho and CONICIT Venezuela (grant S1-9500524).
We are grateful to Dr Theo deVos (SBBI, Seattle) for providing the
genomic L. donovani library, and to Drs Linda Fothergill-Gilmore
(University of Edinburgh) and Fred Opperdoes (ICP, Brussels) for
critical reading of the manuscript.
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