Báo cáo khoa học: 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae Molecular characterization, database searches, modelling studies and evolutionary analysis pptx

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase
in Trypanosomatidae
Molecular characterization, database searches, modelling studies
and evolutionary analysis
Nathalie Chevalier1,*, Luc Bertrand2,3,*, Mark H. Rider2, Fred R. Opperdoes1, Daniel J. Rigden4
and Paul A. M. Michels1
´
1 Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite catholique
de Louvain, Brussels, Belgium
´
2 Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite
catholique de Louvain, Brussels, Belgium
´
3 Division of Cardiology, Universite catholique de Louvain, Brussels, Belgium
4 School of Biological Sciences, University of Liverpool, UK

Keywords
ankyrin-repeat motif; fructose
2,6-bisphosphate; glycolysis regulation;
6-phosphofructo-2-kinase ⁄ fructose-2,6bisphosphatase; trypanosome
Correspondence
P. A. M. Michels, ICP-TROP 74.39, Avenue
Hippocrate 74, B-1200 Brussels, Belgium
Fax: +32 27 62 68 53
Tel: +32 27 64 74 73
E-mail:
*These authors contributed equally to this
work
Database
Nucleotide sequence data are available in
the DDJB ⁄ EMBL ⁄ GenBank databases under

accession numbers AY571277 (Tb4),
AY571278 (Tb1) and AY999068 (Tb2)
(Received 7 April 2005, revised 10 May
2005, accepted 16 May 2005)
doi:10.1111/j.1742-4658.2005.04774.x

Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid
pyruvate kinase and thus represents an important regulator of energy metabolism in these protozoan parasites. A 6-phosphofructo-2-kinase, responsible for the synthesis of this regulator, was highly purified from the
bloodstream form of Trypanosoma brucei and kinetically characterized. By
searching trypanosomatid genome databases, four genes encoding proteins
homologous to the mammalian bifunctional enzyme 6-phosphofructo-2kinase ⁄ fructose-2,6-bisphosphatase (PFK-2 ⁄ FBPase-2) were found for both
T. brucei and the related parasite Leishmania major and four pairs in Trypanosoma cruzi. These genes were predicted to each encode a protein in
which, at most, only a single domain would be active. Two of the T. brucei
proteins showed most conservation in the PFK-2 domain, although one of
them was predicted to be inactive due to substitution of residues responsible for ligating the catalytically essential divalent metal cation; the two
other proteins were most conserved in the FBPase-2 domain. The two
PFK-2-like proteins were expressed in Escherichia coli. Indeed, the first displayed PFK-2 activity with similar kinetic properties to that of the enzyme
purified from T. brucei, whereas no activity was found for the second.
Interestingly, several of the predicted trypanosomatid PFK-2 ⁄ FBPase-2
proteins have long N-terminal extensions. The N-terminal domains of the
two polypeptides with most similarity to mammalian PFK-2s contain a series of tandem repeat ankyrin motifs. In other proteins such motifs are
known to mediate protein–protein interactions. Phylogenetic analysis suggests that the four different PFK-2 ⁄ FBPase-2 isoenzymes found in
Trypanosoma and Leishmania evolved from a single ancestral bifunctional
enzyme within the trypanosomatid lineage. A possible explanation for the
evolution of multiple monofunctional enzymes and for the presence of the
ankyrin-motif repeats in the PFK-2 isoenzymes is presented.

Abbreviations
CDD, conserved domain databases; Fru2,6-P2, fructose 2,6-bisphosphate; FBPase-2, fructose-2,6-bisphosphatase; PEP, phosphoenolpyruvate; PFK-1, 6-phosphofructo-1-kinase; PFK-2, 6-phosphofructo-2-kinase; PKA, protein kinase A; PKC, protein kinase C; TbFBPase-2,
Trypanosoma brucei fructose-2,6-bisphosphatase; TbPFK-2, Trypanosoma brucei 6-phosphofructo-2-kinase.

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PFK-2 ⁄ FBPase-2 of Trypanosomatidae

Fig. 1. Diagrammatic representation of the regulation of carbohydrate metabolism by fructose 2,6-bisphosphate in mammalian cells (A) and
Trypanosomatidae (B). Glycolysis in mammalian cells occurs in the cytosol. In Trypanosomatidae, the glycolytic enzymes responsible for the
conversion of glucose into 3-phosphoglycerate, and the gluconeogenic enzyme fructose-1,6-bisphosphatase are present in glycosomes.
Abbreviations: FBPase-1, fructose-1,6-bisphosphatase; PYK, pyruvate kinase.

Fructose 2,6-bisphosphate (Fru2,6-P2) is a key regulator of glycolysis in almost all eukaryotes, but it is
absent from prokaryotes. In animals, plants and fungi,
this sugar phosphate stimulates glycolysis via allosteric
stimulation of 6-phosphofructo-1-kinase (PFK-1) and
inhibits gluconeogenesis by acting as a negative effector of fructose-1,6-bisphosphatase [1,2] (Fig. 1A). In
contrast, in protozoan organisms belonging to the
Kinetoplastida (comprising pathogenic organisms such
as Trypanosoma and Leishmania) Fru2,6-P2 is not a
stimulator of PFK-1, but rather acts on pyruvate
kinase [3–9]. This latter enzyme is stimulated at submicromolar concentrations, 2000-fold lower than by
fructose 1,6-bisphosphate, the usual regulator of pyruvate kinase activity in other organisms. Similarly, trypanosomatid fructose-1,6-bisphosphatase is insensitive
to Fru2,6-P2. This different enzyme specificity is most
likely related to the unique metabolic regulation in
Kinetoplastida (Fig. 1B). The majority of glycolytic
enzymes responsible for the conversion of glucose into

3-phosphoglycerate are compartmentalized in peroxisome-like organelles called glycosomes [10–12]. Only
the last three enzymes, phosphoglycerate mutase, enolase and pyruvate kinase are present in the cytosol.
The gluconeogenic enzyme fructose-1,6-bisphosphatase
also has a glycosomal localization [12]. Strikingly, glycosomal enzymes such as hexokinase and PFK-1 lack
the regulatory mechanisms found in most other organisms, namely product inhibition and control by metabolites further downstream in the pathway or by
effectors [13]. In Kinetoplastida, such mechanisms
seem to be redundant as a result of the sequestering of
the enzymes within a separate compartment bounded
by a membrane with low permeability to many metabolites [12,14,15]. Interestingly, this compartmentation
FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS

seems to have resulted in a kind of ‘re-routing’ of regulatory mechanisms (Fig. 1) and cytosolic pyruvate
kinase appears the most important regulated enzyme
[5,6,11,12,14].
In mammalian tissues, the bifunctional enzyme
6-phosphofructo-2-kinase (PFK-2; EC 2.7.1.105) ⁄ fructose-2,6-bisphosphatase (FBPase-2; EC 3.1.3.46) catalyses both the synthesis and degradation of Fru2,6-P2
[16,17]. PFK-2 and FBPase-2 activities have also been
detected in Kinetoplastida. In line with the activity
regulation of cytosolic pyruvate kinase by Fru2,6-P2,
the PFK-2 and FBPase-2 activities are localized in the
cytosol of the kinetoplastid Trypanosoma brucei [4].
However, these activities could be separated by partial
protein purification, indicating that they reside in distinct enzymes [4].
In higher animals, different tissue-specific PFK-2 ⁄
FBPase-2 bifunctional isoenzymes exist with kinetic
properties and regulatory mechanisms related to metabolism (glycolysis vs. gluconeogenesis) [1,2,16,17]. The
isoenzymes are homodimers, typically with subunit
masses of 50–60 kDa. They have a common structure,
with the PFK-2 domain comprising most of the N-terminal half of the enzyme subunit and the FBPase-2
domain in the C-terminal half. This central core contains the two catalytic activities and is well conserved.

Both extremities often contain regulatory phosphorylation sites, involved in the fine tuning of enzyme activity. In plants (Arabidopsis, spinach, potato), the
bifunctional enzyme sequence possesses a large N-terminal extension that provides a regulatory domain
[18]. In Saccharomyces cerevisiae, three isoforms of
PFK-2 ⁄ FBPase-2 are present, but each displays only a
single activity [19–21]. Nevertheless, they are clearly
homologous to the mammalian bifunctional enzyme.
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PFK-2 ⁄ FBPase-2 of Trypanosomatidae

In two isoforms (called PFK26 and PFK27), FBPase-2
activity appears to have been lost during evolution as
the result of a crucial substitution or major deletions,
respectively. In the third form (FBP26), the PFK-2
domain has undergone multiple substitutions rendering
it inactive.
For mammalian PFK-2 ⁄ FBPase-2 isoenzymes, it has
been shown that the functional, active form is a dimer.
Although the FBPase-2 domain does not seem to be
involved in dimerization, its presence, whether active
or not, seems to be essential because a mammalian
PFK-2 domain expressed alone in bacteria forms inactive aggregates [22]. By contrast, the bacterially
expressed mammalian FBPase-2 domain is active as a
monomer [23]. The crystal structures of the rat testis
[24] and rat liver bifunctional enzymes [25] have been
solved. The subunits of the dimer are arranged in a
head-to-head fashion with the PFK-2 domains of the
two subunits being intimately associated in both testis
and liver structures. The FBPase-2 domains are independent of each other in the testis enzyme but form

contacts in the liver isozyme. The fact that the PFK-2
domain is structurally related to the adenylate kinase
family, whereas the FBPase-2 domain is similar to
the phosphoglycerate mutase family [24,26], seems to
reveal that the bifunctional enzyme resulted from the
fusion of two ancestral genes.
We studied some properties of PFK-2 (TbPFK-2)
purified from T. brucei as well as bacterially expressed
forms of TbPFK-2 and investigated whether the
T. brucei PFK-2 and FBPase-2 enzymes are homologous to their counterparts in higher eukaryotes.

Results and Discussion
Purification and characterization
of T. brucei PFK-2
PFK-2 was purified from pooled cytosol fractions from
the bloodstream form of T. brucei stock 427 using ionexchange chromatography and specific elution from
Blue Sepharose with buffer containing PFK-2 substrates. The purification was 9000-fold compared with
the activity in the initial extract and the purified
enzyme had a specific activity of 11 mUnitsỈmg)1 of
protein, which is comparable with the specific activity
of PFK-2 in preparations purified from mammalian
tissues [27]. Nevertheless, several bands were visible in
Coomassie Brilliant Blue-stained gels. On gel filtration,
PFK-2 activity eluted from a Superose 12 column as
a single symmetrical peak with a Mr of 76 400 (not
shown). As yeast PFK-2 has been shown to be phosphorylated by protein kinase A (PKA) [28,29] and
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N. Chevalier et al.

Table 1. Kinetic properties of PFK-2 purified from bloodstream-form
T. brucei and effect of phosphorylation by protein kinases. Trypanosome PFK-2 (30 lgỈmL)1) was incubated for 15 min at 30 °C with
or without protein kinases (0.6 unitỈmL)1), purified and assayed as
described previously [87]. Aliquots were taken for PFK-2 activity
measurements. For the fructose 6-phosphate (F6P) saturation
curves, concentrations were varied up to 30 mM. For inhibition by
PEP, the concentrations of fructose 6-phosphate and MgATP were
5 mM. The results are the means ± SEM of three separate determinations, otherwise individual values are given. ND, not determined.

Enzyme

Km F6P
(mM)

Km ATP
(mM)

Vmax
[mUnits
(mg of protein))1]

Untreated
PKA-treated
PKC-treated

5.8 ± 1.2
39
5.8

0.88

ND
ND

7.1 ± 3.2
7.1
6.7

protein kinase C (PKC) [30] with accompanying changes in PFK-2 activity, we tested the effect of phosphorylation by these protein kinases on T. brucei
PFK-2 activity (Table 1). Treatment with PKA led to
PFK-2 inactivation via a sevenfold increase in Km for
fructose 6-phosphate with no change in Vmax, whereas
treatment with PKC was without effect. This contrasts
with yeast PFK-2, in which PKA treatment led to
PFK-2 activation by increasing the Vmax and lowering
the Km for fructose 6-phosphate [28,29] and PKC
treatment led to PFK-2 inactivation [30]. Purified
T. brucei PFK-2 had a pH optimum around 6 (not
shown), was inhibited by phosphoenolpyruvate (PEP;
K0.5 ¼ 0.7 mm) and citrate (60% inhibition at 1 mm)
but like heart PFK-2 [31], was rather insensitive to
inhibition by glycerol 3-phosphate (20% inhibition at
2 mm).
Database searches and sequence analysis
tblastn searches were performed in the databases of
the three trypanosomatid genome projects (T. brucei,
T. cruzi and Leishmania major) using a query of
mammalian and yeast bifunctional PFK-2 ⁄ FBPase-2
sequences. Analysis of the various T. brucei and
L. major databases surprisingly revealed four homologous sequences. The T. brucei sequences and their close
homologues in L. major were named Tb1 ⁄ Lm1,

Tb2 ⁄ Lm2, Tb3 ⁄ Lm3 and Tb4 ⁄ Lm4 and have the database codes shown in Table 2. A diagrammatic representation of the four T. brucei sequences is presented
in Fig. 2. For each of these four isoforms, two corresponding sequences could be found in the T. cruzi
genome database (Table 2), presumably reflecting the
known hybrid genotype of the strain chosen for genome sequencing, as a result of genetic exchange
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N. Chevalier et al.

Table 2. Properties of predicted PFK-2 ⁄ FBPase-2 isoenzymes of trypanosomatids.
Conservation
PFK2-domain

Accession no.
Lm 1
Lm 2
Lm 3
Lm 4
Tb1
Tb2
Tb3
Tb4
Tc1–1
Tc1–2
Tc2–1
Tc2–2
Tc3–1
Tc3–2

Tc4–1
Tc4–2

Chromosome

LmjF3.0800
LmjF26.0310
LmjF36.0150
LmjF07.0760
Tb03.48O8.70
Tb07.27M11.980
Tb10.70.2700
Tb08.29O4.60
Tc00.1047053508153.950
Tc00.1047053508181.20
Tc00.1047053508207.230
Tc00.1047053509509.30
Tc00.1047053510963.50
Tc00.1047053508625.50
Tc00.1047053508569.130
Tc00.1047053503733.20

3
26
36
7
3
7
10
8

FBPase-2 domain

Overallc (%)

Residuesb
[key catalytic
(total 5);
other binding
(total 8)]

Overallc (%)

Presumed
activity

24–40
17–29
9–15
13–25
24–37
13–25
10–16
13–26
23–41
23–41
14–29
14–28
10–15
10–15

13–28
13–27

0;
3;
5;
4;
0;
3;
5;
4;
0;
0;
3;
3;
5;
5;
5;
5;

20–23
24–32
27–36
26–40
18–23
23–29
25–34
30–37
19–25
10–24

24–30
24–29
28–36
28–37
28–40
28–40

PFK-2
PFK-2
FBPase-2
?
PFK-2
?
FBPase-2
?
PFK-2
PFK-2
?
?
FBPase-2
FBPase-2
FBPase-2
FBPase-2

No.
residues

Mass
(kDa)a

Residuesb
[key catalytic
(total 4);
other binding
(total 13)]

2422
667
485
1245
1023
648
478
702
1021
1023
705
702
481
481
749
749

251
74
55
132
111
72
54

79
112
113
79
79
55
54
84
84

4;
4;
1;
3;
4;
2;
0;
2;
4;
4;
3;
3;
0;
0;
2;
2;

13
10
3

6
13
8
4
6
13
13
10
10
3
3
6
6

2
2
8
7
2
2
7
8
2
2
2
2
8
8
7
7

a

Molecular mass calculated from ORF. b Conservation of residues in the predicted trypanosomatid enzymes compared to functional mammalian and S. cerevisiae PFK-2 and FBPase-2 domains (and corresponding to boxed residues in Fig. 3). c Overall percentage of amino acid
sequence identity between the predicted trypanosomatid enzymes and functional PFK-2 and FBPase-2 domains from other eukaryotes.

between two distantly related lineages [32]. Each pair,
for example Tc1–1 and Tc1–2 corresponding to Tb1,
share 95–98% sequence identity overall. Between the
three trypanosomatids, isoenzyme 1 (Tb1, Lm1, Tc1–1
and Tc1–2) representatives share 35–43% sequence
identity. The corresponding figures for isoenzymes 2, 3
and 4 are 43–49, 37–52 and 33–40% (Table 2).

Tb1
Tb2
Tb3
Tb4
500 residues
Fig. 2. Diagrammatic representation of the domain structure of the
various PFK-2 ⁄ FBPase-2 isoenzymes of T. brucei. The domain
structure of the enzymes was inferred from the amino acid
sequences predicted from the ORFs. Light grey, FBPase-2 domain;
dark grey, PFK-2 domain; white, insertions ⁄ extensions compared
with mammalian PFK-2 ⁄ FBPase-2 sequences.

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Mammalian and yeast enzymes usually have molecular masses of 50–60 kDa. By comparison, most of
the trypanosomatidal homologues are atypically large

(Table 2). Most notable are the isoenzymes 1, which
range in size from 1021 residues (112 kDa) for Tc1–1
to 2422 residues (251 kDa) for Lm1. The L. major representative of the isoenzymes 4 is also particularly
large (1241 residues; 132 kDa) compared with the corresponding trypanosomal sequences at around 700–750
residues.
The bulk of known homologues of the bifunctional
enzymes possess both kinase and phosphatase activities. Nevertheless, there is a precedent for homologues
having retained only a single activity in the three yeast
members of the family. In order to predict likely activities for the trypanosomatidal sequences, an analysis
was made of the conservation (or lack of conservation)
of catalytic and substrate binding-site residues in each
domain. For this purpose, we defined sets of key catalytic residues for each of the PFK-2 and FBPase-2
activities (boxed and highlighted in Fig. 3): nonconservative replacement of any of these residues would
be expected to abolish activity. For PFK-2 activity key
catalytic residues were Lys51, Thr52, Asp128 and
Lys172. Site-directed mutation of these residues
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N. Chevalier et al.

A

B

Fig. 3. Sequence alignments of the kinase domain (A) and bisphosphatase domain (B). In each case, the trypanosomatid sequences are
compared with domains of confirmed activity, rat testis bifunctional enzyme (PDB code 2BIF) [24] in both cases, and the respective monofunctional S. cerevisiae enzymes [SWISSPROT codes 6P21_YEAST in (A) and F26_YEAST in (B)]. Numbers substitute large insertions to the
rat testis enzyme. For clarity, only one of each pair of T. cruzi homologues is shown. Rat testis enzyme numbering is shown beneath the

alignment. Key catalytic residues and additional binding residues are boxed, with the former also shown emboldened and italicized. Within
each box shading is used for functional conservation of the particular residue, i.e. as an indication that the residue present would have the
same capacity for electrostatic interaction, hydrogen bonding or hydrophobic interaction, as the residue present in rat testis enzyme. The
figures were produced with ALSCRIPT [88].

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N. Chevalier et al.

drastically reduces activity [33–35]. Similarly, at the
FBPase-2 site, Arg255, His256, Arg305, Glu325 and
His390 were considered to be key residues [36–38]. We
also considered other residues involved in substrate
binding (boxed in Fig. 3) based mainly on X-ray crystal structures. The binding of ATP analogues in the
PFK-2 active site and of fructose 6-phosphate and
inorganic phosphate ions to the FBPase-2 active site
have been visualized crystallographically [24,25,39].
The binding site for fructose 6-phosphate in the
PFK-2 catalytic site has yet to be directly visualized,
but conserved residues in the vicinity have been the
subject of several docking and site-directed mutagenesis studies enabling modelling of fructose 6-phosphate
binding [26,35,40–42].
Striking differences in the patterns of conservation of
these residues were immediately apparent. For isoenzyme 1, all key kinase catalytic residues were conserved
and additional substrate-binding residues were also well
conserved (Fig. 3A; Table 2). In sharp contrast, no
FBPase-2 key catalytic residues were conserved

(Fig. 3B; Table 2). For example, His256, which is transiently phosphorylated during the catalytic cycle of
FBPase-2, is replaced by proline in all isoenzyme 1
sequences. Comparisons of whole domains tell a similar
tale – the isoenzyme 1 kinase domains are 23–41% identical to presumed active PFK-2 domains, whereas the
corresponding range for the bisphosphatase domains is
18–25%. Similarly, the isoenzyme 1 sequences are better
conserved in their N-terminal domains (59–68%
sequence identity, excluding the comparison of Tc1–1
and Tc1–2) than in their C-terminal domains (40–59%).
These data strongly suggest that Tb1, Lm1 and Tc1
homologues are monofunctional PFK-2s.
Lm2 also conserves the key catalytic kinase residues
and none of the changes in other substrate binding
residues seems incompatible with kinase activity,
although Asn63 and Arg193, predicted to hydrogen
bond to fructose 6-phosphate, are replaced by Phe
and Val, respectively. Surprisingly, the trypanosomal
representatives of isoenzyme 2 have nonconservative
replacements in the key kinase residues; Tb2 has Met
and Ala for Thr52 and Asp128, whereas both T. cruzi
sequences replace Asp128 with Asn (Fig. 3A; Table 2).
Given the key role of Asp128 in coordination to the
catalytically essential divalent metal cation [24], these
substitutions appear to rule out kinase activity for
Tb2, Tc1–1 and Tc1–2. Indeed, additional substrate
binding residues are less well conserved in these
sequences. At the FBPase-2 site, both key catalytic
residues and additional substrate-binding residues are
poorly conserved (Fig. 3B; Table 2). In particular, the
replacements of His256 and Glu325 rule out FBPase-2

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PFK-2 ⁄ FBPase-2 of Trypanosomatidae

activity for isoenzyme 2. Comparisons of whole
domain conservation are uninformative: the kinase
domains of the isoenzymes 2 are 13–29% identical to
active kinase domains, whereas their bisphosphatase
domains are 22–32% identical to other bisphosphatase
domains. Surprisingly, given the patterns of residue
conservation, the C-terminal domain is slightly better
conserved between isoenzyme 1 sequences (58–65%,
again excluding the comparison of Tc2–1 and Tc2–2)
than the N-terminal domain (47–58%). Taken
together, the data suggest that Lm2 probably has
monofunctional kinase activity but that experimental
characterization would be needed to confirm the prediction. Tb2, Tc1–1 and Tc1–2 are predicted, surprisingly, to have neither kinase nor FBPase-2 activity.
For isoenzyme 3, clear-cut predictions may once
again be made. In the kinase domain, key catalytic residues are absent from all sequences and other substrate-binding residues are not conserved (Fig. 3A). At
the FBPase-2 catalytic site, all residues are very highly
conserved in all four sequences. Similarly, the kinase
domains of isoenzyme 3 sequences are just 9–15%
identical with active domain sequences, whereas the
bisphosphatase domain is well conserved at 25–36%
identity. Also, the C-terminal domain is much better
conserved in an intertrypanosomatid comparison
(62–73%) than the N-terminal domain (16–34%). Thus
Lm3, Tb3, Tc3–1 and Tc3–2 would clearly be monofunctional FBPase-2 enzymes.
In the kinase domain of isoenzyme 4, the replacement of Asp128 with Asn in all four sequences, as well
as various substitutions of Lys172, rules out kinase

activity. Additional substrate-binding residues are also
poorly conserved (Fig. 3A; Table 2). Isoenzyme 4
sequences are also much better conserved, compared
with active domain homologues, in the bisphosphatase
domain than in the kinase domain. Conservation in
the bisphosphatase domain is in the range 26–40%
compared with just 13–28% in the kinase domain. The
corresponding figures for the intertrypanosomatid isoenzyme 4 comparison are 47–59% for the bisphosphatase domain and 34–53% identity in the kinase
domain. The T. cruzi sequences have all the required
FBPase-2 key catalytic residues and well-conserved
additional substrate-binding residues. The other two
isoenzyme 4 sequences have nonconservative replacements of key catalytic residues; the substitution of
Arg255 by Leu in Lm4 and the replacement of His256
by Asn in Tb4. These argue against their having
FBPase-2 activity, but in each case mutations elsewhere in the catalytic site make it difficult to completely rule out activity. The loss of Arg255, a residue
that binds the 2-phospho group of substrate and the
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phosphohistidine intermediate [37,43], could be partially compensated by the presence of neighboring
His416 (replacing Leu in rat testis) which could form
an ionic interaction with the 2-phosphate group.
Equally, the lack of phosphorylable His256 in Tb4
would typically be thought to be sufficient to abolish
bisphosphatase activity. However, experiments on
FBPase-2 and relatives show that caution should be
exercised. Most importantly, when this His was
replaced by Ala in the rat testis enzyme, a surprising

17% of catalytic activity was maintained [44], probably
due to water taking over the nucleophilic role [39]. It
is also relevant to note the surprising variations in key
catalytic residues in members of the related phosphoglycerate mutase superfamily [45]. In the case of Tb4,
it is also intriguing to note the replacement of Asn262
(in rat testis enzyme) with an additional His at position 262. The new His is well placed to interact with
the 2-phosphate group of the incoming substrate.
However, it seems unlikely that this new His may take
over the role of the missing His256 to form the phospho-enzyme intermediate, as it is not suitably positioned for in-line attack on the 2-phosphate group of
the substrate. In summary, although Tc4–1 and Tc4–2
are probably monofunctional bisphosphatases, experimental data would be required to test the possibility of
Lm4 and Tb4 sharing the same function.
Heterologous expression, purification and
characterization of T. brucei PFK-2/FBPase-2
We set out to test experimentally the results of the bioinformatics analysis of sequences retrieved from the
databases. To that end, PCR amplification was first
performed for Tb1 and Tb4 fragments, using as
template genomic DNA from our laboratory strain,
T. brucei stock 427. The fragments thus obtained were
used to screen an available genomic library. DNA
fragments were subcloned in plasmids and sequenced.
All clones obtained contained either of the two distinct
genes. The Tb1 and Tb4 sequences in the database of
T. brucei stock TREU927 ⁄ 4 have the same length as
the proteins encoded by the genes analysed by us for
T. brucei stock 427 (Table 2), but differ by a number
of substitutions. For Tb1, substitutions were found at
five positions (Thr18Met, Pro51Leu, His384Arg,
Ala422Gly and Lys630Glu). Only the latter substitution, corresponding to position 138 in the rat testis
enzyme [24], is within the PFK-2 domain, but the residue is not part of the active site. The other four positions lie in the N-terminal extension. The Tb4 amino

acid sequences of stocks TREU927 ⁄ 4 and 427 differ
at 12 positions by single amino acid changes; two
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N. Chevalier et al.

substitutions (Ser–Asn at position 536 of the fulllength predicted protein and His–Gln at position 589)
are within the PFK-2 ⁄ FBPase-2-specific region, but are
not expected to have any consequence for enzyme
activity. The first of these positions is on an insertion
relative to the rat testis enzyme and the second corresponds to position 347. These differences should, most
likely, be attributed to polymorphisms between the
T. brucei strains used by us and in the genome-sequencing project.
For Tb2, the full-length gene of stock 427 was
amplified and sequenced. No differences were found
between the Tb2 nucleotide sequences of the two
stocks.
To prove the identity of Tb1 as an active PFK-2,
and to confirm that the Tb2 is inactive as predicted,
we expressed the proteins in a heterologous system.
However, it was anticipated that the large Tb1 polypeptide as predicted from the full-length open reading
frame (ORF), would be difficult to express as a soluble
active protein. Therefore, a shorter part comprising
the region homologous to the bifunctional PFK2 ⁄ FBPase-2 enzymes of higher eukaryotes was chosen
for expression. A protein starting at codon ATG 505
(giving the N-terminal sequence MSSSYTTVSDAVSL-)
corresponds quite well with the beginning of the structurally resolved part of the rat testis bifunctional
enzyme and from where good alignment is possible
(the multiple alignment in Fig. 3A starts with the
underlined last three residues). Moreover, this protein

still contains the region corresponding to the N-terminal part of other PFK-2s that is involved in dimerization. The shorter ORF codes for a polypeptide of
519 amino acids (including the initiator methionine),
with a calculated molecular mass of 57 078 Da and a
pI value of 9.29. When expressed with a His-tag, as
described in Experimental procedures, a protein of 547
residues with a predicted molecular mass of 60 306 Da
and a pI of 9.29 is produced. Under rather specific
growth conditions, adapted from Oza et al. [46], low
amounts of soluble enzyme could be obtained that
indeed displayed PFK-2 activity. The protein was partially purified. On SDS ⁄ PAGE several bands were
visible, but the identity of a polypeptide of Mr
 60 000 was confirmed as Tb1 after western blotting
and immunodetection with anti-(poly His) sera (not
shown). Expression of larger constructs was also
attempted, both using Escherichia coli cells and
in vitro, in a coupled transcription–translation system
(Rapid Translation System, Roche Molecular Biochemicals), using different vector systems, differently
placed tags for affinity purification, and a variety of
conditions for bacterial growth and induction of
FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS


N. Chevalier et al.

protein expression. However, in most cases very poor
expression of an inactive, unstable protein was
obtained, or the protein was expressed as insoluble
inactive enzyme.
The active 60 000 Mr Tb1 was subjected to a preliminary kinetic analysis. The K mapp for fructose 6-phosphate varied between 1.9 ± 0.12 and 4.6 ± 0.8 mm,
whereas the K mapp for ATP was between 1.6 ± 0.27

and 2.0 ± 0.30 mm in four different preparations of
enzyme. These values are similar to those for the
enzyme partially purified from bloodstream-form trypanosomes (see above) and to values reported previously [4]. With regard to their PFK-2 activity, the
various mammalian isoenzymes display much lower
Km values: 15–150 times for fructose 6-phosphate and
3–20 times for ATP [4,16].
The relatively good conservation of the ATP-binding
site residues between mammalian and Tb1 proteins
suggests that the explanation for the lower ATP affinity of the latter must lie with the three significantly different positions, residue 220 (Val in mammalian
enzymes, Lm1 and Tc1, but Ala in Tb1), residue 246
(Val or Ile in mammalian enzymes, Pro in the trypanosomatid proteins) and position 427 (Tyr in mammalian enzymes, Gly, Glu and Asp in Lm1, Tc1 and Tb1,
respectively). The branched side chains of residues 220
and 246 form the side of the adenine-binding pocket
further away from the catalytic site and each make
multiple hydrophobic interactions with the heterocyclic
ring. Their replacement with nonbranched Ala and Pro
would reduce the steric complementarity of adenine
and its pocket, thereby reducing the strength of the
interaction. Also, the hydrogen bond from Tyr427 to
the a-phosphate of ATP is not present in Tb1. Instead,
the replacement Asp could, assuming local correctness
of the sequence alignment, lead to electrostatic repulsion of the negatively charged phosphate groups of
ATP.
Bacterially expressed Tb1 was analysed by gel filtration over a Superdex 200 HR 10 ⁄ 30 column, to determine its oligomeric state. However, under all
conditions tested (various buffers, variable ionic
strength, presence of reducing agents – Experimental
procedures) all Tb1, as detected by western blotting
using an antiserum specific for the His6-tag, eluted as
an entity of high mass (> 600 kDa) with a low PFK-2
activity. In addition, a second protein peak with higher

PFK-2 activity eluted with a relative molecular mass
of  140 kDa, suggesting it was a dimer. Purified rat
liver PFK-2 ⁄ FBPase-2, used as a control, eluted as a
110 kDa dimer as detected by both PFK-2 activity and
western blotting using a homologous antiserum. These
results suggest that the bacterially produced Tb1
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PFK-2 ⁄ FBPase-2 of Trypanosomatidae

enzyme is an active dimer that has a strong tendency
to aggregate.
In contrast to Tb1, no activity was found for bacterially expressed, soluble Tb2, in agreement with the
predictions of the sequence analysis. Surprising in this
respect is that the Tb2 gene sequences found in the
two different T. brucei stocks were identical and that
an Expressed Sequence Tag corresponding to Tb2 has
been found in procyclic T. brucei rhodesiense libraries
(GenBank accession no. AA689209.1). This suggests
that this protein, despite its lack of PFK-2 activity,
may play a role in these trypanosomes.
The fact that only Tb1 displays activity, and that no
other sequences with typical PFK-2 features could be
detected in any of the trypanosomatid databases,
strongly suggests that the 76.4 kDa protein purified
from trypanosomes is a Tb1 form, despite the fact that
the complete ORF of Tb1 gene codes for a 110 kDa
polypeptide. We hypothesize, therefore, that the purified protein represents a processed form of the protein
(see also below). Future studies will also include a
detailed experimental analysis of the two T. brucei proteins with likely and possible FBPase-2 activity, Tb3

and Tb4, respectively. A Tb4 construct of stock 427
trypanosomes has already been expressed in E. coli,
although so far in mainly an insoluble form, and Tb3
will be expressed in the future. FBPase-2 assays were
not performed for Tb1 and Tb2, as it is inconceivable
that these proteins would possess any FBPase-2 activity, as explained above (also Table 2).
Evolution of PFK-2/FBPase-2
The amino acid sequences of the bifunctional PFK-2 ⁄
FBPase-2 enzymes from many organisms were
retrieved from the swissprot database, aligned with
those of the various trypanosomatid PFK-2 ⁄ FBPase-2
homologues and used for evolutionary analysis. All the
PFK-2 ⁄ FBPase-2 sequences can be conveniently divided into an N-terminal PFK-2 domain and a C-terminal FBPase-2 domain. In the yeast PFK27 sequence,
the FBPase-2 domain is difficult to recognize. Presumably its sequence diverged considerably and was truncated during evolution. The PFK-2 domain is related
to a family of nucleotide-binding proteins including
adenylate kinase, p21 ras, EF-Tu, the mitochondrial
ATPase b-subunit and myosin ATPase, all of which
have a similar fold and contain the Walker A and B
motifs. The FBPase-2 domain also belongs to a protein
family comprising the cofactor-dependent phosphoglycerate mutases and acid phosphatases. The bifunctional PFK-2 ⁄ FBPase-2 must have originated by fusion
of representatives of these two families in a common
3549


PFK-2 ⁄ FBPase-2 of Trypanosomatidae

ancestor of all eukaryotic organisms studied here (trypanosomatids, yeasts and fungi, plants and animals).
The PFK-2 and FBPase-2 domains can be flanked by
extensions of variable lengths. In plants, Neurospora
crassa and yeast PFK26 the N-terminal extensions can

be long and increase the molecular mass from  55 to
90 kDa. From the ORFs, we infer that in some trypanosomatid isoenzymes, the N-terminal extensions
can be even longer. The extremities of mammalian isoenzymes serve as regulatory domains often containing
phosphorylation sites. Moreover, it has been shown
that the N-terminal domain of the Arabidopsis enzyme
is important both for subunit assembly and for defining the kinetic properties of the enzyme [47]. In each
of the yeast and trypanosomatid isoenzymes, one of
the catalytic cores seems to have been inactivated, rendering the bifunctional enzyme monofunctional.
The sequences of PFK-2 ⁄ FBPase-2 in animals and
plants form distinct clusters in phylogenetic trees made
separately for the PFK-2 and FBPase-2 domains, with
adenylate kinase and phosphoglycerate mutase as outgroups, respectively (Fig. 4). In plants, only a single
gene of the bifunctional enzyme was detected [18,48],

N. Chevalier et al.

whereas animals have different bifunctional isoenzymes, represented by four subtrees (corresponding to
the liver ⁄ muscle, heart ⁄ kidney, testis and brain ⁄
placenta groups, respectively). The evolution of the
PFK-2 ⁄ FBPase-2 in the lineages of yeasts ⁄ fungi and
Trypanosomatidae is more difficult to deduce from the
phylogenetic trees. This is due to: (a) the relatively low
level of conservation of these sequences, and the longer
evolutionary distances when these organisms are considered; and (b) the fact that the inactivated domains
of these enzymes have possibly been subject to a very
high evolution rate. For the same reason, the highly
aberrant C-terminal domain of S. cerevisiae PFK27
was omitted from the FBPase-2 tree. Nevertheless, our
preliminary analysis suggests that most isoenzymes of
the fungi result from gene duplications within this

group. Furthermore, in the FBPase-2 domain tree, all
the Trypanosomatidae sequences are together in one
cluster separated from the sequences of all other
organisms (but containing the outgroup). The isoenzymes form individual groups. Isoenzymes 1 and 2,
containing all the putative PFK-2s (Lm1, Tb1, Tc1–1,
Tc1–2 and Lm2), cluster together, as do isoenzymes 3

Fig. 4. Phylogenetic trees of the PFK-2 and FBPase-2 domains of both the bifunctional and monofunctional proteins. All PFK-2 and ⁄ or
FBPase-2 containing sequences from animals, invertebrates, fungi and protists, as obtained from the SWISSPROT ⁄ TREMBL databases
(Experimental procedures) were aligned with each other using CLUSTALX [80]. From this alignment subalignments were created containing
either the PFK-2 domain or the FBPase-2 domain. Each of the subalignments was used for the creation of a neighbour-joining tree from a
matrix of uncorrected pair-wise distances using the tree option within CLUSTALX. Regions with insertions or deletions were excluded from the
analyses. Horizontal bars represent 10 substitutions per 100 residues. The trees were rooted using either an Arabidopsis thaliana chloroplast
adenylate kinase or E. coli cofactor-dependent phosphoglycerate mutase as an outgroup.

3550

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N. Chevalier et al.

and 4, containing all the likely FBPase-2s (Lm3, Tb3,
Tc3–1, Tc3–2, Tc4–1 and Tc4–2). The situation for the
trypanosomatids in the PFK-2 tree is very similar,
except also for the presence of the S. cerevisiae PFK27
in the cluster. Furthermore, the phylogenetic analysis
also showed that the formation of the four isoenzymes
in the trypanosomatids has occurred already in the
common ancestor of the genera Trypanosoma and

Leishmania.
The presence of multiple isoforms of PFK-2 ⁄
FBPase-2 in mammals can be understood as a need
for distinct enzymes each with different kinetic properties and regulatory mechanisms optimized in regulating
glycolysis and ⁄ or gluconeogenesis in the different tissues [1,16]. With regard to yeast, the two isoenzymes
with PFK-2 activity differ in that only PFK26 is activated by protein phosphorylation [29], whereas the
synthesis of PFK27 is only induced by fermentable
carbon sources [21]. However, the growth rates and
glycolytic flux of both the PFK26 and PFK27 deletion
mutants of S. cerevisiae are similar to that of wild-type
cells [19,21], and did not reveal an essential role of
Fru2,6-P2 in the regulation of carbon fluxes in this
organism [49]. Nor could different roles for the two
PFK-2s be demonstrated by metabolome analysis of
the mutants [50].
Our data do not permit us to draw any conclusions
as to the reason why trypanosomatids have four isoenzymes. Sequence inspection and structure modelling
suggested that some of them (Tb1, Lm1, Tc1–1,
Tc1–2 and Lm2) are monofunctional PFK-2s,
whereas others (Tb3, Lm3, Tc3–1, Tc3–2, Tc4–1 and
Tc4–2) most likely only have FBPase-2 activity. The
sequence analysis suggested that these proteins are all
soluble. It did not reveal obvious topogenic signals
indicative for functioning of isoenzymes in different
cell compartments. Only Tb2, the inactive PFK-2 of
T. brucei, contains a potential peroxisome-targeting
signal at its C-terminus (-NKL) [51], but a similar
tripeptide was not found on the corresponding
sequences of the other trypanosomatids. It could be
imagined that isoenzymes with different properties are

necessary at different stages of the life cycle of these
organisms. Many trypanosomatid species are pathogenic organisms with a highly complicated life cycle.
T. brucei cycles between the mammalian bloodstream,
the tsetse fly midgut and the insect’s salivary gland.
These are radically different environments where the
parasite encounters different nutrients and has to
adapt its metabolism accordingly. Leishmania species
undergo similar transitions between flies and mammals where they predominantly live intracellularly in
the phagolysosomes of macrophages.
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PFK-2 ⁄ FBPase-2 of Trypanosomatidae

Why the bifunctional PFK-2 ⁄ FBPase-2 evolved into
different monofunctional enzymes in the yeasts and,
most likely, also in trypanosomatids is not clear. Possibly, it represents an as yet not understood adaptation
to the specific requirements of glucose metabolism in
these unicellular organisms, different from the requirements in the ancestral eukaryote where the fusion of
the PFK-2 and FBPase-2 domains in a single enzyme
occurred and different from that in extant animals and
plants with their bifunctional enzymes. Usually, opposite metabolic reactions are catalysed by separate
enzymes; PFK-2 ⁄ FBPase-2 is an exception. Moreover,
bifunctionality extends to its substrate ⁄ product,
Fru2,6-P2, which in higher eukaryotes (but not in trypanosomatids) has two targets, PFK-1 and FBPase-1.
The advantages of the association of opposite reactions may be: (a) simplicity in short-term control, such
as regulation at a single site by an allosteric effector or
phosphorylation; and (b) simplicity of long-term regulation (one gene, one mRNA). One possible reason
why several microorganisms have, at a later stage of
evolution, again uncoupled the PFK-2 and FBPase-2
activities is that it may have endowed an increased

flexibility to adapt to different growth conditions: different combinations of monofunctional PFK-2 and
FBPase-2 enzymes may be expressed in cells growing
in different environments. However, this remains to be
studied by following the expression of the different
enzymes throughout the life cycle of the trypanosomatids. That each of the monofunctional enzymes retained
(part of) the inactivated domain is in line with the
notion that both domains may be required for proper
folding or oligomerization.
The inferred activities of some trypanosomatid isoenzymes remain to be confirmed. Enzymes with similar
activities may differ in kinetic and regulatory properties. We have demonstrated that Tb1 has PFK-2 activity, whereas no activity could be found for Tb2, in
agreement with sequence analysis predictions. The
apparent Km values of Tb1 for fructose 6-phosphate
and ATP are similar to those of the enzyme partially
purified from the bloodstream-form trypanosomes
(Table 1) [4]. The lack of FBPase-2 activity in this
enzyme is highly likely. It should be noted, however,
that we do not know if the absence of N-terminal
domains may have affected the activity of the bacterially expressed enzyme.
It is interesting to note that Expressed Sequence
Tags corresponding to Tb2 (GenBank accession no.
AA689209.1; unpublished data) and Tb3 (T26149) [52]
have been obtained from procyclic T. brucei rhodesiense libraries. Our unpublished data (N. Chevalier and
P. A. M. Michels, unpublished) showing the presence
3551


PFK-2 ⁄ FBPase-2 of Trypanosomatidae

of Tb1 protein during the same procyclic stage, suggest
that proteins with opposite catalytic activities (Tb1 is a

PFK-2; Tb3 is strongly predicted to have FBPase-2
activity) are simultaneously present during the trypanosomatid life cycle, reinforcing the notion that the trypanosomatid enzymes should be subject to regulation.
Indeed, we have shown that the activity of PFK-2
purified from trypanosomes can be regulated by phosphorylation.
Conserved motifs in the N-terminal regions
of trypanosomatid PFK-2s
The ORFs of trypanosomatid isoenzymes 1, 2 and 4
extend well upstream of the region corresponding to
the mammalian bifunctional enzymes (Fig. 2). The predicted amino acid sequences contain normal amounts
of predicted regular secondary structure. Except for a
small region (see below), no obvious homology with
other sequences in databases was observed. In the gene
sequences, no trypanosomatid-specific motifs indicative
of RNA trans-splicing [53,54] could be found. Cissplicing is very rare in these organisms and only one
example has been reported to date [55]. The possibility
of splicing was further explored. To that end, PCR
experiments were performed with different sets of
primers, covering the various parts of the Tb1
(TbPFK-2) ORF, using as a template cDNA prepared
with oligo-dT as primer on total RNA from the cultured bloodstream form of T. brucei. The results confirmed that the entire ORF was present as a single
mRNA (not shown).
Among the trypanosomatid sequences, the upstream
regions are not conserved either, except for an
approximately 135-residue stretch that could be detected in the putative PFK-2s – isoenzymes 1 and 2.
Searches in primary and secondary structure databases
revealed the presence of two ankyrin repeats within
this region. For example, an e-value of 1e)16 was
obtained for entry cd00204, comprising four ankyrin
repeats, when searching in the Conserved Domain
Databases (CDD) with the Lm1 N-terminal extension

sequence. Only 60% of the cd00204 entry was matched
to the Lm1 sequence and searches with the other trypanosomal homologues in secondary sequence databases also gave hits for just two ankyrin repeats.
Because small numbers of consecutive ankyrin repeats
are rare, apparently for stability reasons [56], we carried out more sensitive fold recognition experiments.
With the prekinase domain portions of isoenzymes 1
and 2, more ankyrin repeats were revealed, both within
the conserved region shared by the four sequences and
in the remaining portions not apparently homologous
3552

N. Chevalier et al.

between the sequences of isoenzymes 1 and 2.
Although the fold-recognition scores were highly significant for various ankyrin repeat structures in the
PDB, differences in the extent of matches, resulting in
different predicted numbers of ankyrin repeats, were
evident in the results of different fold-recognition
methods. However, these uncertainties were confined
to the apparently nonhomologous parts of the prekinase domain regions. In the conserved region, there was
an excellent match between predicted secondary structure and actual secondary structure of a designed artificial ankyrin repeat (PDB code 1MJ0) [57], for
example (Fig. 5). It should be remembered that the
characteristic b-turns of the ankyrin repeat structure
(shown as pairs of arrows in Fig. 5) are not predicted
by 3-state secondary structure prediction programs.
With the exception of T. cruzi isoenzyme 2 sequences,
the conserved region clearly contains four ankyrin
repeats. In Tc2–1 and Tc2–2, a deletion compared with
the other trypanosomatid sequences is evident, corresponding exactly to a whole ankyrin repeat. The b-turn
prior to the first helix pair is not present, but this is
often the case for ankyrin repeat structures including

the designed ankyrin protein included in the alignment.
The pairwise sequence identity between Lm1, Tb1 and
Tc1–1 in this region is between 60 and 65%, whereas
Lm2, Tb and Tc1–2 share 27–43% identity. The two
groups share 23–29% identity between them, whereas
the trypanosomal sequences share 26–33% sequence
identity with the designed ankyrin repeat protein [57].
Outside the portions shown in Fig. 5, additional
ankyrin repeats are present in each of the four homologues (Fig. 6). Although the sequences seem to
diverge more from the standard ankyrin repeat consensus, fold recognition alignments and secondary structure predictions of characteristic pairs of helices (as in
Fig. 5) provide strong evidence for extra repeats. In
Lm1, up to seven repeats may be present immediately
following the conserved region in Fig. 5. However, this
still leaves around 500 residues after the extra repeats
before the start of the kinase domain, for which no
structure may be proposed. In the cases of Tc1–1,
Tc1–2 and Tb1, there is evidence for three ankyrin
repeats before the conserved region and the same number following. For Lm2 and Tb2, there appears to be
a single additional ankyrin repeat before the conserved
region and another immediately following (Fig. 6).
Tc2–1 and Tc2–2 have a single clear ankyrin repeat
following those shown in Fig. 6. These different numbers of ankyrin repeats most likely reflect the results of
domain duplication and ⁄ or deletion. The results (not
shown) of the radar repeat detection program [58]
for Tc1–1, Tb1 and Lm2 suggest that the third and
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PFK-2 ⁄ FBPase-2 of Trypanosomatidae

N. Chevalier et al.

Fig. 5. Ankyrin motifs in trypanosomatid PFK-2 ⁄ FBPase-2 sequences. Alignment of the four ankyrin repeats in the conserved upstream
region of the putative trypanosomal PFK-2 sequences with the designed ankyrin repeat protein (1MJO) of Kohl et al. [57]. For clarity, only
one of each pair of T. cruzi homologues is shown. Regular secondary structure predicted by PSIPRED [79] is shown above the alignment for
the L. major sequences and the true secondary structure of 1MJO below the alignment. Cylinders represent a helices, whereas pairs of
arrows are used for the b turns. Shading marks positions where the residue of the trypanosomal homologue is identical to that of the crystal
structure and positions well conserved among the homologues are emboldened. The figure was produced with ALSCRIPT [88].

fourth repeats in Fig. 5 arose through a duplication.
Similarly, the two repeats predicted to immediately
precede the conserved region in Tb1 and Tc1 have
similarity detectable by radar. The same program
suggests that the region beginning with the clear ankyrin repeats shown in Fig. 5 and extending to the start
of the kinase domain in Lm1 may consist largely of
five highly diverged duplicated sequences. The first of
these copies likely contains six ankyrin repeats, the last
three of the four shown in Fig. 6 along with three
more. Although the clear independent evidence for
ankyrin repeats in the second copy supports the idea
Tb1
Lm1
Tc1_1
Tb2
Lm2
Tc2_1
500 residues

Fig. 6. Diagrammatic representation of ankyrin-motif repeats in
Tb1, Lm1, Tc1–1, Tb2, Lm2 and Tc2–1. PFK-2 and FBPase-2

domains in the C-terminal halves of the proteins are indicated as in
Fig. 2. In the N-terminal halves, the ankyrin motifs with a primary
structure having high similarity to the consensus motifs are indicated in darker grey than the motifs showing less conservation of the
primary structure, but having a repeat length and predicted secondary structure that conform to the consensus.

FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS

of the duplication, the alignment of the five copies
reveals only poor sequence conservation overall so that
a definitive conclusion is impossible.
The ankyrin repeat is a protein sequence motif that
is widespread in nature. It is found in proteins from
bacteria, fungi, plants and animals, and is present in
proteins at different locations of the cell as well as in
secreted proteins. Its function is to mediate protein–
protein interactions. Within a protein, the motif can
form distinct units of 2–20 consecutive stacking repeats
with a hydrophobic core. The units thus form scaffolds
with a stability depending on the number of repeats
and displaying a variable surface, each optimized to
interact specifically with one out of a wide range of
other, unrelated nonankyrin repeat-containing macromolecules [56,57,59]. The trypanosomatid PFK-2 molecules seem to be produced as long molecules with a
variable number of authentic ankyrin-motif repeats in
their N-terminal extensions. It also seems likely that
in these trypanosomatid proteins, the ankyrin repeat
structures are involved in establishing interactions
between proteins. However, it remains to be determined
with which proteins. Known interaction partners of
homologues from other species include other enzymes
of sugar metabolism – glucokinase [60] and PFK-1 [61]

– but also 14-3-3s proteins whose binding to cardiac
[62] and plant [63] enzymes regulates their activity.
Other attractive candidate partners for the trypanoso3553


PFK-2 ⁄ FBPase-2 of Trypanosomatidae

N. Chevalier et al.

matid enzymes are the (putative) FBPase-2s, the isoenzyme 4 sequences, which have a long N-terminal
extension that may contain a binding site for the PFK-2
ankyrin repeats. However, there is no detectable similarity between the N-terminal extensions of Lm4 and
Tb4. The notion of a noncovalent interaction between
distinct PFK-2 isoenzymes and a FBPase-2 would combine the advantages of both the flexibility offered by
different combinations of multiple isoenzymes and the
formation of a bifunctional (in this case heteromeric)
enzyme, as discussed above. However, alternative functions for the ankyrin repeat motif cannot be excluded.
For example: (a) mediating the interaction of PFK-2
with other proteins in the cell, such as cytoskeleton
constituents or membrane proteins; or (b) targeting the
PFK-2 precursor protein to a possible macromolecular
complex for processing. Further research is required to
unravel the complex picture of the PFK-2 ⁄ FBPase-2
isoenzymes in the Trypanosomatidae.

cose 6-phosphate before loading a DEAE–Sepharose column
(5 · 6.5 cm). After extensive washing with 20 mm Hepes
pH 7.5, 50 mm KCl, 2.5 mm MgCl2, 0.5 mm EDTA,
0.25 mm EGTA, 1 mm benzamidine ⁄ HCl, 20% (v ⁄ v) glycerol, 15 mm 2-mercaptoethanol, 5 mm potassium phosphate, 0.2 mm phenylmethanesulfonyl fluoride, 1 lgỈmL)1
antipain, 0.1 mm fructose 6-phosphate and 0.3 mm glucose 6-phosphate (buffer A), the column was eluted in a linear salt gradient (0–0.75 m KCl) in 500 mL buffer A.

Fractions (10 mL) were pooled, diluted with an equal volume
of buffer A and applied to a column of Blue Sepharose
(1.5 · 5 cm) equilibrated with buffer A. After washing overnight with 500 mL buffer A supplemented with 0.2 m KCl,
the enzyme was eluted in buffer A supplemented with 0.18 m
KCl and 1 mm MgATP. Fractions containing activity were
pooled, concentrated by ultrafiltration, dialysed overnight
against 500 mL of buffer A and stored in aliquots at )80 °C.

Conclusions

Encouraged by the near-complete status of various trypanosomatid genome projects, we carried out searches in trypanosomatid genome databases. These employed the blast
[65] facilities of the GeneDB resource (edb.
org) [66] which unites nucleic acid and protein sequence
information obtained from the T. brucei and L. major
genome projects and a separate resource (http://www.
TcruziDB.org/) [67] for T. cruzi. Sequences of confirmed
PFK-2 ⁄ FBPase-2 homologues were obtained from the
enzyme database [68] and supplemented with others seen in
blast searches of the nr database [69]. These homologues
are all presumed to be bifunctional with the exception
of the three yeast enzymes [19–21]. Searches in secondary sequence databases were carried out at the CDD
( [70].
Fold recognition experiments employed metaserver
( [71] and their results were
interpreted in the light of the Livebench benchmarking
effort ( [72]. Sequences
were aligned using t-coffee [73] or muscle [74] and the
resulting alignments hand-edited and manipulated using
jalview [75]. modeller [76] was used to calculate percentage identities between sequences, using the formula number
of identities divided by the length of the shorter sequence

[77]. The likely structural and functional effects of sequence
differences between the trypanosomal and mammalian
homologues were envisaged using the available crystal
structures and the program o [78]. Protein secondary structure predictions were made with psipred [79].
For a phylogenetic analysis, sequences for most of the
organisms were taken from the SWISSPROT database
(6P21_YEAST, 6P22_YEAST, F261_BOVIN, F261_
HUMAN, F261_RAT, F262_ARATH, F262_BOVIN,
F262_HUMAN, F262_MOUSE, F262_RAT, F262_

Unexpectedly, trypanosomatids have been shown to
contain four genes for PFK-2 ⁄ FBPase-2 isoenzymes,
or four pairs in the case of T. cruzi. Mirroring the situation in S. cerevisiae, each of the four trypanosomal
homologues, which seem to have arisen through trypanosomatid lineage-specific duplications, is predicted
to be, at most, monofunctional. It seems likely that
expression of multiple monofunctional enzymes offers
additional flexibility of metabolic regulation in the
trypanosomes whose life cycles involve dramatic environmental shifts. One T. brucei homologue was demonstrated to possess PFK-2 activity when expressed in
E. coli. Its kinetic parameters match those of PFK-2
purified from bloodstream-form T. brucei, which is
regulated by phosphorylation as seen in other species.
Most surprisingly, all predicted kinases contain ankyrin repeats in long, variably sized N-terminal extensions, which presumably interact with protein targets
yet to be identified.

Experimental procedures
Purification of PFK-2 from T. brucei
Bloodstream-form trypomastigotes of T. brucei stock 427
were grown in rats and purified, and lysates prepared as described previously [64]. To a high-speed supernatant fraction
(1500 mL) from  200 g wet weight of trypanosomes was
added 15 mm 2-mercaptoethanol, 5 mm potassium phosphate, 0.2 mm phenylmethanesulfonyl fluoride, 1 lgỈmL)1

antipain, 0.1 mm fructose 6-phosphate and 0.3 mm glu-

3554

Database searches, sequence alignment,
structure modelling and phylogenetic analysis

FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS


N. Chevalier et al.

SOLTU, F262_SPIOL, F263_BOVIN, F263_HUMAN,
F263_RAT, F264_HUMAN, F264_RAT, F26L_CHICK,
F26 °CAEEL, F26_YEAST) and for Schizosaccharomyces
pombe (Q8TFH0 and T50154) and Neurospora crassa
(Q9P522) from the TREMBL database. All sequences were
aligned together with the trypanosomatid sequences using
clustalx [80]. Adenylate kinase (KADC_ARATH) and cofactor-dependent phosphoglycerate mutase (GPMB_ECOLI)
were added as outgroups for the PFK-2 and FBPase-2
domains, respectively. The two separate domains were used
for the creation of phylogenetic trees using the tree option of
clustalx after exclusion of positions with gaps. Owing to
the low degree of some of the pairwise identities between
sequences, no correction for multiple substitution could be
applied. Therefore, branch lengths represent observed distance percentages between sequences rather than evolutionary distances.

Construction of expression clones, production
and purification of recombinant T. brucei PFK-2
Fragments of two potential PFK-2 ⁄ FBPase-2 sequences

(denoted Tb1 and Tb4, see Results and Discussion section),
as recognized in the databases of the T. brucei (stock
TREU927 ⁄ 4) genome project, were amplified on T. brucei
stock 427 genomic DNA and used as a radioactively
labelled hybridization probe to screen a genomic library of
this strain prepared in E. coli with the phage vector
kGEM11 (Promega, Madison, WI) [81]. Plaques of positive
clones were processed, the DNA was purified and appropriate restriction fragments containing the PFK-2 and
FBPase-2 genes were subcloned in plasmid vectors. Each
part of the genes was sequenced at least once in both directions, using a Beckman CEQ 2000 sequencer (Beckman
Instruments, Fullerton, CA, USA). A third gene, coding
for the protein Tb2, was amplified over its full length,
cloned and sequenced.
In order to express Tb1 in E. coli, different parts of the
ORF, each with a different potential start codon but the
same stop codon (Results and Discussion) were amplified
by PCR, using a sense oligonucleotide containing a NdeI
restriction site just upstream of the chosen start codon and
an antisense oligonucleotide with an XhoI site immediately
before the PFK-2 stop codon. The PCR products of the
expected size were purified and ligated into the pGEM-T
Easy vector (Promega). After checking their sequence, the
amplified fragments were excised from the recombinant
plasmid by digestion with NdeI and XhoI and ligated in the
expression plasmid pET28a (Novagen, Inc., Madison, WI,
USA), digested with the same enzymes. E. coli BL21(DE3)
cells were transfected with these constructs. Each plasmid
directs the synthesis of a Tb1 with a 20-amino acid N-terminal extension including six adjacent His residues (‘Histag’) and an eight-residue C-terminal extension, also having
a His6-tag. To develop a Tb2 expression construct, a gene-

FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS

PFK-2 ⁄ FBPase-2 of Trypanosomatidae

internal NdeI site was mutated first by introducing a silent,
single-nucleotide substitution (using the QuickChange protocol of Stratagene). Subsequently, the amplified full-length
gene, with NdeI and BamHI sites at the start and stop
codon positions, was ligated in the corresponding sites of
pET28.
Cells harbouring a recombinant plasmid Tb1 or Tb2
were grown at 37 °C and 250 r.p.m. in 500 mL of Terrific
Broth medium [82] supplemented with 30 lgỈmL)1 kanamycin. When the culture reached an D600 of  1.4, the temperature was reduced to 30 °C, agitation was reduced to
100 r.p.m. and isopropyl thio-b-d-galactoside was added to
a final concentration of 0.5 mm to induce the expression of
the protein and growth was continued for about 16 h. Cells
were collected by centrifugation (3000 g, 15 min, 4 °C) and
resuspended in 30 mL of cell lysis buffer containing 20 mm
Tris ⁄ HCl, pH 8, 0.5 m NaCl, 5 mm MgCl2, 0.03% (w ⁄ v)
Brij35, 0.1 mm fructose 6-phosphate, 0.3 mm glucose
6-phosphate and a protease inhibitor mixture (Roche
Molecular Biochemicals, Mannheim, Germany). Cells were
lysed by two passages through a SLM-Aminco French pressure cell (SLM Instruments, Inc., Urbana, IL, USA) at
90 MPa. Nucleic acids were degraded by treatment with
125 units of Benzonase (Merck, Germany) for 10 min at
37 °C. The lysate was centrifuged (12 000 g, 10 min, 4 °C)
and TbPFK-2 was purified from the soluble cell fraction by
metal-affinity chromatography (TALON resin, BD Biosciences-Clontech, Franklin lakes, NJ, USA). Briefly, 2 mL
of resin was added to the suspension and mixed for 20 min
at room temperature. The resin with bound protein was
washed twice for 10 min with 20 mL lysis buffer (with centrifugation at 700 g, 5 min), transferred to a column and

washed twice again with 5 mL lysis buffer supplemented
with 5 and 10 mm imidazole, respectively. Finally, protein
was eluted with 10 mL lysis buffer containing 50 mm
imidazole and 1-mL fractions were collected. EDTA and
dithiothreitol were immediately added to each fraction at
concentrations of 2.5 and 5 mm, respectively.

Protein measurements, SDS/PAGE, western
blotting, gel filtration
Protein concentrations were determined using the Bio-Rad
(Hercules, CA) protein assay, based on the Bradford
Coomassie Brilliant Blue-binding procedure [83], using
bovine serum albumin as a standard.
SDS ⁄ PAGE was carried out by the Laemmli method
[84]. After electrophoresis, gels were either stained with
Coomassie Brilliant Blue, or used for immunoblotting
according to the method of Towbin [85]. The membranes
(polyvinylidene difluoride membrane, Roche Molecular Biochemicals) were blocked by incubation in phosphatebuffered saline (NaCl ⁄ Pi) containing 0.1% Tween 20 and
5% (w ⁄ v) low-fat milk powder. For detection of the protein, the primary antibody (monoclonal anti-His serum,

3555


PFK-2 ⁄ FBPase-2 of Trypanosomatidae

Amersham Biosciences, Amersham, UK) was diluted
(1 : 15,000–25 000) in NaCl ⁄ Pi containing 0.5% milk powder. The secondary antibody, anti-(mouse IgG) conjugated
to horseradish peroxidase (Rockland Immunochemicals,
Inc., Gilbertsville, PA, USA), was diluted 1 : 40 000 and
visualized with the ECL Western Blotting System, a

luminol-based system (Amersham Biosciences).
The native molecular mass of TbPFK-2 was determined
by gel filtration. Protein purified from parasites (0.15 mL)
was loaded on a Superose 12 column (Amersham Biosciences) equilibrated in 50 mm Hepes pH 7.5, 100 mm KCl,
0.1 mm EDTA, 1 mm dithiothreitol, 1 mm potassium phosphate, 0.1 mm fructose 6-phosphate and 0.3 mm glucose 6-phosphate. Fractions (0.2 mL) were collected at
a flow rate of 0.3 mLỈmin)1 and assayed for PFK-2
activity. The native molecular mass of the bacterially
expressed form of TbPFK-2 ⁄ FBPase-2 with PFK-2 activity
(Tb1) was determined using a Superdex 200 HR 10 ⁄ 30 column (Amersham Biosciences) equilibrated in a buffer specified below. A Tb1 preparation (2 mL), eluted from the
TALON resin and concentrated to 0.25 mL, was loaded
onto the column and eluted at a flow rate of 0.4 mLỈmin)1
using the equilibration buffer. Fractions of 0.4 mL were
collected and the presence of Tb1 was determined by enzymatic PFK-2 assays and western blotting. The columns
were calibrated with gel filtration standards from Bio-Rad
ranging from 1350 to 670 000 Da, and purified rat liver
PFK-2 ⁄ FBPase-2. For the recombinant enzyme, gel-filtration experiments were performed with various protein
batches prepared in different ways. When the protein had
been purified under the standard conditions, using a
Tris ⁄ HCl buffer, 0.5 m NaCl and various additions as described above, the equilibration buffer used contained
20 mm Tris ⁄ HCl, pH 8, 0.5 m NaCl, 5 mm MgCl2, 0.03%
(w ⁄ v) Brij35, 10% (v ⁄ v) glycerol, 0.1 mm fructose 6-phosphate, 0.3 mm glucose 6-phosphate, a protease inhibitor
mixture, with or without 15 mm 2-mercaptoethanol. Other
batches of protein had been prepared specifically for the
gel-filtration experiments with a buffer containing 50 mm
Hepes ⁄ KOH, pH 7.5, 250 mm KCl, 0.03% (w ⁄ v) Brij35,
0.1 mm fructose 6-phosphate, 0.3 mm glucose 6-phosphate,
50 mm imidazole, 2.5 mm EDTA, 5 mm dithiothreitol and
a protease inhibitor mixture. In these cases, gel filtration
was performed with 50 mm Hepes ⁄ KOH, pH 7.5, 100 mm
KCl, 0.03% (w ⁄ v) Brij35, 10% (v ⁄ v) glycerol, 5 mm

EDTA, 0.1 mm fructose 6-phosphate, 0.3 mm glucose
6-phosphate, 15 mm 2-mercaptoethanol and a protease
inhibitor mixture.

Enzyme assay and kinetic studies
PFK-2 activity was assayed in buffer containing 50 mm
Tris ⁄ HCl at pH 7.1, 5 mm potassium phosphate, 1 mm
dithiothreitol, 100 mm KCl, 20 mm KF, 1 mgỈmL)1 of
albumin and appropriate concentrations of substrates as

3556

N. Chevalier et al.

previously described [86]. For Km measurements, the concentrations of substrate were up to 10 times the Km value.
For fructose 6-phosphate saturation curves, the concentration of MgATP was 10 mm. For MgATP saturation curves,
the concentration of fructose 6-phosphate was 10 mm. Kinetic constants were calculated by fitting the data to a hyperbola by nonlinear least-squares regression using the
sigmaplot computer package. Auxiliary enzymes (aldolase,
triosephosphate isomerase and glycerol-3-phosphate dehydrogenase) and cofactors (ATP, NADH) were from Roche
Molecular Biochemicals.

Acknowledgements
This research was supported by grants from the European Commission through its INCO-DEV programme
(contract ICA4-CT-2001–10075) to PM and DR,
´
the Belgian ‘Fonds de la Recherche Scientifique Medicale’ (FRSM) to PM, and the Belgian Interuniversity
Attraction Poles – Federal Office for Scientific, Technical and Cultural Affairs to FO. LB was supported by
´
the ‘Actions de Recherche Concertees’ 98 ⁄ 03-216 from
the French Community of Belgium and is currently a

Research Associate of the ‘Fonds National de la
Recherche Scientifique’ (Belgium). We thank Prof
Louis Hue (ICP, Brussels) for many stimulating discussions and his critical reading of the manuscript and
Mr Rudi Parrado Vargas for his contribution to some
experiments.

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