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Eur. J. Biochem.

265, 10151021 (1999) q FEBS 1999

Characterization and molecular cloning of an adenosine kinase from


Babesia canis rossi
Celine Carret1, Stephane Delbecq1, Gilles Labesse2, Bernard Carcy1, Eric Precigout1, Karina Moubri1,
Theo P. M. Schetters3 and Andre Gorenflot1
1

Laboratoire de Biologie Cellulaire et Moleculaire, EA MESR 2413, UFR des Sciences Pharmaceutiques et Biologiques, Montpellier, France;
Centre de Biochimie Structurale, CNRS UMR C9955, INSERM U414, Universite Montpellier I, France; 3Department of Parasitology,
Intervet International BV, Boxmeer, the Netherlands

In the search for immunoprotective antigens of the intraerythrocytic Babesia canis rossi parasite, a new cDNA
was cloned and sequenced. Protein sequence database searches suggested that the 41-kDa protein belongs to the
phosphofructokinase B type family (PFK-B). However, because of the low level sequence identity (, 20%) of
the protein both with adenosine and sugar kinases from this family, its structural and functional features were
further investigated using molecular modelling and enzymatic assays. The sequence/structure comparison of the
protein with the crystal structure of a member of the PFK-B family, Escherichia coli ribokinase (EcRK),
suggested that it might also form a stable and active dimer and revealed conservation of the ATP-binding site.
However, residues specifically involved in the ribose-binding sites in the EcRK sequence (S and N) were
substituted in its sequence (by H and M, respectively), and were suspected of binding adenosine compounds
rather than sugar ones. Enzymatic assays using a purified glutathione S-transferase fusion protein revealed that
this protein exhibits rapid catalysis of the phosphorylation of adenosine with an apparent Km value of 70 nm,
whereas it was inactive on ribose or other carbohydrates. As enzymatic assays confirmed the results of the
structure/function analysis indicating a preferential specificity towards adenosine compounds, this new protein of
the PFK-B family corresponds to an adenosine kinase from B. canis rossi. It was named BcrAK.
Keywords: adenosine kinase; glutathione S-transferase fusion protein; homology modelling; intracellular parasite;
structure/function analysis.

Adenosine kinase (ATP : adenosine 5 0 -phosphotransferase,


EC 2.7.1.20) catalyses the phosphorylation of adenosine,
using ATP as the main phosphoryl donor in the presence of
magnesium, to release AMP and ADP. Most studies on this
enzyme have been directed towards designing inhibitors,
especially in parasitic protozoa and humans. In humans, it
has been suggested that inhibitors of adenosine kinase could
play an important pharmacological role in increasing intravascular adenosine concentrations and act as anti-inflammatory
agents [1,2]. In parasitic protozoa (e.g. Babesia, Plasmodium,
Toxoplasma, Leishmania, Trypanosoma), it has been shown that
Correspondence to B. Carcy, Laboratoire de Biologie Cellulaire et
Moleculaire, EA MESR 2413, UFR des Sciences Pharmaceutiques et
Biologiques, 15 Avenue Charles Flahault, F-34060 Montpellier Cedex 2,
France. Fax: + 33 0 4 67 54 66 21, Tel.: + 33 0 4 67 54 64 81,
E-mail: bcarcy@ww3.pharma.univ-montp1.fr
Abbreviations: BcrAK, B. canis rossi adenosine kinase; EcRK, E. coli
ribokinase; IPTG, isopropyl thio-b-d-galactoside; GST, glutathione
S-transferase.
Enzymes: adenosine kinase (gene adk, EC 2.7.1.20); fructokinase
(gene scrK, EC 2.7.1.4); ribokinase (gene rbsK, EC 2.7.1.15);
1-phosphofructokinase (gene fruK, EC 2.7.1.56); 6-phosphofructokinase
(gene pfkB, EC 2.7.1.11).
Note: the one-letter amino acid code is used. Nucleotide sequence data
reported in this paper are available in the EMBL, GenBankTM and DDBJ
databases under accession number AJ223322.
Note: web site available at http://www.cbs.univ-montp1.fr/,labesse/ak.html
(Received 27 May 1999, revised 3 August 1999, accepted 23 August 1999)

these parasites lack the ability to synthesize purine nucleotides


de novo and are dependent on their host for these key
metabolites [1]. Adenosine kinase is involved in the purine
salvage pathway which is considered to be essential as it is an
effective way of blocking these parasites [2,3]. Thus, understanding the catalytic mechanism of adenosine kinase could be
important both from the fundamental point of view and in the
hope that a detailed knowledge of the enzyme may provide
relevant information necessary to design specific inhibitors.
Adenosine kinases have been purified from a number of
eukaryotic sources, including human placenta and liver [4,5],
rabbit liver [6], yeast [7] and Leishmania donovani [4], and
extensively characterized at a kinetic level. Important results
have been achieved for the leishmanial enzyme, demonstrating
that it displays many specific characteristics compared with
other adenosine kinases including that from the natural host of
this parasite [4,5,8]. Such data are of great use in the design of
specific parasitic inhibitors. Moreover, until now, the only
information on the active-site structure of adenosine kinases
was obtained from this leishmanial adenosine kinase using a
chemical-modification approach [9,10]. These studies suggested the existence of two discrete catalytically active
nucleoside-interacting sites. A comparative analysis with the
purified homologue from hamster, a natural host of L. donovani, demonstrated the presence of a specific arginine residue
at or near the adenosine-binding site of the leishmanial
enzyme [9].
However, despite these studies, the fine structure/function of
adenosine kinase has not been investigated using sequence

1016 C. Carret et al. (Eur. J. Biochem. 265)

analysis. Adenosine kinase from humans [11,12], rats [11], the


plant Physcomitrella [13], yeasts (unpublished results, direct
submission to the EMBL/GenBank/DDBJ databases under
accession numbers P47143 and 3184089), the nematode
Caenorhabditis elegans [14] and L. donovani (unpublished
results, direct submission to EMBL under accession number
S52758) were sequenced. Sequence analysis has shown that
adenosine kinase has similarities to microbial ribokinases and
fructokinases [12] from the phosphofructokinase B type
(PFK-B) family [15]. The latter kinases were grouped together
because they all share an original ATP-binding site characterized by two specific consensus regions [15]. The recently
solved crystal structure of the Escherichia coli ribokinase
(EcRK) combined with ADP and ribose has revealed the
structural basis of specific ATP binding as well as the particular
substrate-recognition site [16]. This opens the way for a better
analysis of the specificities and biochemical features of PFK-B
enzymes at the molecular level. However, despite the common
ATP-binding site, these kinases have different functions and
share a low overall sequence identity (around 20%), preventing
precise determination of the functional feature of a new member.
Indeed, while usually straightforward in cases of high
sequence identity (over 35%), the alignment of all the
sequences belonging to the same structural family with
conventional homology search programs [17,18] becomes
more uncertain and tedious at low sequence identity level
(below 25%) [19]. Additional methods for the correct gathering
and alignment of all the sequences are available for the analysis
of a new protein sequence belonging to a given structural
family [2022]. Furthermore, the correct conservation of the
three-dimensional structures may lead to identification of a
distantly related homolog from an already determined threedimensional structure [23,24]. Such an approach, with a careful
analysis of the deduced structural model, has been shown to be
helpful for determination of specific functional features in spite
of low sequence identity [2528]. Thus, by combining both a
refined alignment and a three-dimensional structure analysis,
comparative molecular modelling may facilitate the experimental characterization of new homologues.
Here, we report on the structure/function analysis of an
adenosine kinase at the molecular level. The corresponding
cDNA was cloned and sequenced from Babesia canis rossi, an
intraerythrocytic protozoan parasite responsible for canine
babesiosis, a malaria-like tick-transmitted disease, in SouthAfrica. Protein sequence database searches [17,18] revealed
that the encoded protein belonged to the PFK-B family [15] but
could not identify its precise function (, 20% amino acid
identity). The function of this new protein was investigated
using both comparative molecular modelling with the crystal
structure of EcRK [16] and enzymatic assays with a purified
glutathione S-transferase (GST) fusion protein. Both
approaches indicate an adenosine kinase specificity for this
novel PFK-B protein, hereafter named BcrAK (B. canis rossi
adenosine kinase).

M AT E R I A L S A N D M E T H O D S
Construction of the cDNA library
Total RNA and mRNA of B. canis rossi were isolated using,
respectively, the RNAgentsw Total RNA Isolation System and
the PolyATtract mRNA Isolation Systemw III as described by
the manufacturer (Promega). Poly(A) (5 mg) was converted
into double-stranded cDNA and used to construct the cDNA
library with the ZAP ExpressTM cDNA Gigapackw II Gold

q FEBS 1999

Cloning Kit (Stratagene). After the ligation of an EcoRI linker


and the release of an XhoI restriction site, the size-fractionated
cDNA (from 500 to 4000 bp) was ligated to the EcoRI/XhoI cut
phosphatase-treated lZAPII vector. The resulting DNA was
packaged in vitro using Gigapackw II Gold extracts (Stratagene) and introduced into E. coli XL1-blue. The primary
library was amplified in the same bacterial strain and the
quality of the library was estimated by performing a blue/white
color selection using 0.5 m isopropyl thio-b-d-galactoside
(IPTG) and 25 mgmL21 5-bromo-4-chloroindolyl-b-dgalactopyranoside according to the manufacturer's instructions.
Screening of the cDNA library
About 105 clones were plated on to E. coli XL1-blue and
allowed to grow overnight at 37 8C. Nitrocellulose filters
(Amersham), soaked in 10 mm IPTG and air-dried, were placed
on the plates and the cultures were incubated for 3 h at 37 8C.
A 100-fold dilution of a hyperimmune serum (collected from a
dog repeatedly infected with B. canis rossi parasites) depleted
of anti-(E. coli) antibodies and a 500-fold dilution of
peroxidase-conjugated goat anti-(dog IgG) (Sigma) were used
for the cDNA library screening. Immunopositive clones were
detected using 4-chloro-1-naphthol as chromogen. Positive
clones were purified by two additional cycles.
Isolation of plasmid DNA
The pBK-CMV plasmids were excised in vitro as described by
the manufacturer using R408 helper phage (Stratagene).
JetQuick spin columns were used to prepare the selected
recombinant plasmid according to the manufacturer's instructions (Genomed, Oeyenhausen, Germany). The size of the
insert was checked by double digestion with EcoRI/XhoI and
electrophoresis on a 0.8% agarose gel.
DNA sequencing
Nucleotide sequence was determined by the dideoxy chain
termination method from alkali-denatured double-strand
templates [29] on both strands of the selected plasmid using
the T7 Sequencing Kit (Pharmacia Biotech) and ([a-35S]thio)dATP (Amersham). Primers were T7 and T3 universal primers
(Stratagene) and four 20-mer oligonucleotides derived from the
established sequence of each strand.
Protein sequence comparison and molecular modelling
Protein sequence database searches were performed with the
blast [17] and psiblast version 2.0.5 programs [18] with
default parameters. Each blast or psiblast search output was
analysed as a single multiple alignment by filtering significant
pairwise alignments using mulblast [21]. Alignment refinement was subsequently performed using the program tito [24]
where the EcRK was used as a template [16]. Threedimensional models were built using modeller 4.0 [30] and
assessed using prosa 1.0 [31] and verify3D [32]. These threedimensional structures were visualized on a UNIX workstation
using xmmol [33]. The BcrAK secondary structures (a-helix,
b-strand and coil) were assigned using the program psea [34].
Pairwise and multiple alignments were confirmed by hydrophobic cluster analysis as previously described [20], in order to
delineate the structurally conserved regions along the amino
acid sequences of BcrAK and EcRK.

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Molecular cloning of a B. canis rossi adenosine kinase (Eur. J. Biochem. 265) 1017

Expression and purification of cloned BcrAK in E. coli


The EcoRI/XhoI fragment containing the complete encoding
amino acid sequence of the cDNA BcrAK was excised from
the pBK-CMV recombinant plasmid and subcloned into the
EcoRI/XhoI cut phosphatase-treated pGEX 4T-3 prokaryotic
vector (Pharmacia Biotech). The E. coli BL21 cells were
transformed with the ligation mixture and positive clones were
selected using restriction enzyme digestion analysis. Expression and purification of the recombinant fusion GSTBcrAK
were performed as previously described [35] with minor
modifications for the expression step. The BL21 colony
positive for the recombinant BcrAK cDNA was grown in
2YT medium (16 gL21 bactotryptone, 10 gL21 bacto-yeast
extract, 5 gL21 NaCl, pH 70; Life Technologies Inc.) at 37 8C
until the culture reached an A600 of 1.2. Before the overexpression of the GSTBcrAK, the culture was heat shocked
for 30 min at 42 8C. The GSTBcrAK overexpression was
then induced by 0.1 mm IPTG for 2 h at 37 8C. SDS/PAGE
(12% gel) was used to control the overexpression, the
molecular mass and the purity of the GSTBcrAK.
Enzymatic assays with the GSTBcrAK
Enzymatic activity of the GSTBcrAK was investigated by a
standard radiochemical assay. It was prepared in a final volume
of 15 mL, which contained 50 mm Tris/HCl (pH 7.5), 5 mm
MgCl2, 0.2 mm [g-32P]ATP (specific activity 3000 Cimmol21;
Amersham Pharmacia Biotech), 1 mm appropriate substrate
(adenosine or carbohydrates such as ribose, fructose and
fructose 6-phosphate) and 5 mg GSTBcrAK. As a control,
the same experiments were performed with 5 mg of native GST.
Reaction mixtures were incubated for 10 min at 37 8C, and
aliquots of 1 mL from each tube were then spotted on to
polyethyleneimine-cellulose TLC plates (POLYGRAMw CEL
300 UV254; Macherey-Nagel) which were eluted for 60 min.
For the adenosine assay, elution was performed by a twofold
development in acetic acid (1 m) followed by development in
LiCl2 (1.2 m) to top off the sheet. For the carbohydrate assays, it
was performed by twofold development with ethyl acetate/
pyridine/water (2 : 1 : 2, v/v). The TLC plates spotted with
adenosine sample were dried and placed in autoradiography
cassettes. The 32 P-labelled nucleotides were visualized by film
exposure at 280 8C for 10 min. The TLC plates spotted with
carbohydrate samples were dried and detected after being
sprayed with anisidine phthalate reagent ( p-anisidine
12.3 gL21 and phthalic acid 16.6 gL21 in 95% ethanol). A
phosphoimager (Molecular Dynamics) was used to determine
the amount of AMP or phosphoryl sugar formed. The 32Plabelled product of the BcrAK catalysis in the presence of
adenosine was identified as AMP by loading non-radioactive
AMP and ADP on the same plate, and then visualized by
UV254. Adenosine (or 2 0 -deoxyadenosine) disappearance was
followed similarly (data not shown).

R E S U LT S A N D D I S C U S S I O N
Isolation and sequence of the BcrAK cDNA
Although effective chemotherapeutic treatment of infected
animals is available, the prevention of babesiosis by vaccination
seems to be a great opportunity especially using soluble
parasite antigens as vaccine [36]. Moreover, whatever the
species studied, soluble parasite antigens with molecular mass
around 40 kDa, seem to play an important role in the
development of efficient vaccine [37,38]. In an effort to

identify such antigens of B. canis rossi, a cDNA library


consisting of 7.5  106 p.f.u.mL21 containing 5% nonrecombinant clones was screened with hyperimmune serum
collected from a dog repeatedly infected with B. canis rossi
parasites. Among the several positive clones purified, a cDNA
encoding a 41-kDa protein was sequenced and studied further.
The complete nucleotide and amino acid sequences of the
presumed adenosine from B. canis rossi (BcrAK) (see below)
are available in the EMBL, GenBankTM and DDBJ databases
under accession number AJ223322. The BcrAK cDNA
contained 1246 nucleotides with a 1104-bp open reading
frame. It encodes a protein of 368 residues with an expected
molecular mass of 40.8 kDa, in agreement with molecular mass
data obtained for previously described adenosine kinase
proteins [4,1113]. A specific feature of BcrAK is the
presence, at its N-terminus, of a strongly hydrophobic stretch
of 20 residues which could correspond to a signal peptide with
a predicted cleavage site between the A20 and the Y21 residues
[39], suggesting that BcrAK might be secreted. Although it
remains to be demonstrated experimentally, it would be an
original feature, as secreted adenosine kinase has not been
reported previously.
Protein sequence comparison
Sequence database screening using the BcrAK sequence as a
query was performed with both blast and psiblast programs
[17,18]. It indicated distant homologies with various enzymes
belonging to the PFK-B family. With the blast program, these
distantly related proteins were mainly of bacterial origin with
probability scores as low as 1.2 e-14 (where e is defined by the
blast program as statistical significance) for the first hit (an
uncharacterized carbohydrate kinase from Synechocystis sp.), in
the range of e-10 to e-4 for different sugar kinases, and a
probability score of 0.00019 for the first adenosine kinase, the
pairwise matches clustering at the specific consensus region of
the ATP-binding site. At the convergence of the psiblast
database screening (after 13 iterations), the mammalian
adenosine kinases were the first hits (probability scores e-65
to e-60; sequence identity 16%) with the longest pairwise
alignments (over 350 amino acids). Various bacterial sugar
kinases followed immediately after (probability scores e-59 to
e-50), despite a slightly higher sequence identity (up to 18%
over 310 amino acids). Among the consensus motifs highlighted by mulblast filtering, BcrAK possesses two specific
motifs (PROSITE PS00583 and PS00584 [40]; in BcrAK
residues 88112 and 305318) confirming its classification in
the PFK-B family [15]. However, the precise function of the
newly cloned protein remained unclear, as the low sequence
identity scores (less than 20%) and the puzzling order of the
homologues according to their origin and their substrate
specificity were not conclusive.
Structure/function relationship
To ensure the proper alignment of the BcrAK sequence with the
other PFK-B proteins, a previously described strategy was
applied [24]. This allows a rapid and simultaneous analysis of a
multiple sequence alignment, and it measures the compatibility
of the aligned sequences with a known three-dimensional
structure. The recently solved crystal structure of EcRK [16]
was used as a template despite the low sequence identity (18%
over 340 amino acids). This dimeric ribokinase is composed of
a main domain comprising the active site and the substrate, and
the ATP-binding site as observed in the crystallized ternary
complex [16]. Its C-terminus comprises the subdomain

1018 C. Carret et al. (Eur. J. Biochem. 265)

q FEBS 1999

Fig. 1. Sequence/structure alignment of BcrAK and three known PFK-B enzymes. The multiple sequence alignment including the fructokinase and
ribokinase from E. coli (ScrK, accession number P40713 and EcRK, accession number P05054, respectively), the BcrAK (accession number AJ223322), and
the human adenosine kinase (hAK, accession number P55263), was deduced from the structure/sequence comparison performed using the program tito.
Conserved motifs involved in the ATP binding are labelled ATP1, ATP2 and ATP3 as described in the text. Similarly the motifs lying in the substrate binding
pocket are labelled as SUB and the position interacting with the 2 0 -hydroxyl group is marked by a star. Identities are shown on a black background, and
similarities on a grey background. The putative signal peptide is marked by a hatched bar and the dimerization subdomain is underlined. The secondary
structures (a19 and b19) were predicted by similarity with those assigned in the known crystal structure of the ribokinase (EcRK) and they were labelled
to highlight the domain organization as well as the relationship of the catalytic domain with the Rossmann fold. The BcrAK sequence numbering is indicated
above the alignment.

involved in the phosphate donor binding. The ribose-binding


site and part of the active site is covered by a small antiparallel
b sheet protruding from the N-terminus subdomain and
involved in the dimerization. tito pseudo-energy [24] validated
the proposed alignment (Fig. 1) of the BcrAK sequence and the
EcRK structure (E total, 277.5 per aligned residues; E total is
the pseudo-energy based on the aligned 3D structure). Based on
the refined alignment, a three-dimensional model was built
using modeller 4.0 [30] and checked using the programs
prosa 1.0 [31] and verify3D [32]. The global pseudo-energy
(residues 1350) in prosa is 20.7 kcal (versus 22.0 for the
crystal EcRK structure) and the score in verify3D reaches
< 0.2 (versus < 0.6 for the crystal EcRK structure). These
values suggest, in spite of the low sequence identity level, that
the proposed fold is correct.
Moreover, hydrophobic cluster analysis [20] suggested the
conservation of the same fold for both the BcrAK and the
EcRK molecules (hydrophobic cluster analysis alignment is
available at http://www.cbs.univ-montp1.fr/,labesse/ak.html),
derived from the so-called Rossmann fold [41].
From the sequence/structure comparison of BcrAK with
EcRK, the residues lying in the ATP-binding site were strictly
conserved and clustered in three main sequence motifs at
the C-terminus of BcrAK. The motif 306TxxxGDxF313
(designated ATP3 in Fig. 1) corresponds to the signature
PROSITE PS00584. Two additional motifs 240TxNxxE245
and 279TxG281 (ATP1 and ATP2, respectively, in Fig. 1) were

derived from the sequence/structure comparison. The conservation of both motifs and the surrounding secondary structures
suggests that BcrAK and EcRK interact with their common
phosphorus donor, in the same way.
From this structural comparison, the main change in the
kinase structure corresponds to the region comprising the
substrate-binding site (designated SUB in Fig. 1). This change
includes particular residue substitutions as well as a 15 amino
acids long insertion in the BcrAK. In the current model this
extra sequence stretch points toward the active site of the
second monomer in the dimeric enzyme. This suggests that it
might be involved in adenine moiety recognition. Two specific
amino acid substitutions were highlited by the analysis of
ribose binding in the EcRK crystal structure. Residues H41 and
M43 in BcrAK (boldface type in Fig. 1) correspond in EcRK to
the S12 and N14 which interact with the sugar. In particular, the
larger and hydrophobic side chain of M43 would not allow the
ribose to bind in the same conformation in BcrAK as the N14
allows in EcRK. The particular conformation of ribose
complexed to EcRK remains unclear [16]. However, the
distinct ribose conformation observed in the adenosine
compounds in various protein complexes might explain the
discrimination between the ribose alone and the same sugar
attached to a purine. Further analysis revealed that the other
adenosine kinase as well as the fructokinases have in common a
valine on the position equivalent to that occupied by the
methionine M43 in BcrAK (indicated with a star in Fig. 1).

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Molecular cloning of a B. canis rossi adenosine kinase (Eur. J. Biochem. 265) 1019

Fig. 2. Overexpression and purification of the GSTBcrAK from


E. coli. Protein was purified from BL21 cells after a 2-h IPTG induction
as described, separated on a SDS/12% polyacrylamide gel, and stained with
Coomassie brilliant blue. Lanes: 1, E. coli lysate; 2, lysate of E. coli
containing the recombinant pGEX 4T-3/BcrAK induced by IPTG; 3,
purified GSTBcrAK; M, prestained protein molecular mass markers
(BRL). The arrow indicates the position corresponding to the 60-kDa
BcrAK protein.

This suggests that adenosine and fructose might bind in the


substrate pocket with their 2 0 -hydroxyl group in an orientation
differing greatly from that of the ribose alone. However, the
adenosine kinase might discriminate fructose from nucleoside
by specific interactions with the base moiety. These latter
interactions would also repel the entrance of phosphorylated
sugars such as fructose 6-phosphate and fructose 1-phosphate
because of their strongly negative charge. The adenosine kinase
might also possess a higher affinity for 2 0 -deoxyribosecontaining compounds. Such a distinct affinity has been
measured previously for human adenosine kinase [12]. In
contrast, the 3 0 -hydroxyl group is hydrogen-bonded to a strictly
conserved aspartate (D45 in BcrAK, bold-faced in Fig. 1) and
is predicted to be essential for the correct orientation of a
substrate in the active-site pocket.
Expression of the GSTBcrAK and enzymatic assays
To verify that the cDNA clone-encoded BcrAK contained
adenosine kinase activity rather than sugar kinase activity, the
entire coding sequence of BcrAK was subcloned into the
prokaryotic expression vector pGEX. Overexpression of the
GSTBcrAK fusion protein was performed in the BL21 E. coli
strain. Expression studies demonstrate that E. coli culture

Fig. 3. Adenosine kinase activity of the


GSTBcrAK. (A) TLC detection was used for
the determination of enzymatic activity: 1 mL of
each reaction mixture was spotted on the
POLYGRAM CEL 300 UV254 plaque
(Macherey-Nagel), dried, and eluted as
described. Lanes: 1, 1 mm adenosine, 0.2 mm
[g-32P]ATP, 5 mm MgCl2 and 5 mg native GST,
after a 10-min incubation at 37 8C; 2, 1 mm
adenosine, 0.2 mm [g-32P]ATP, 5 mm MgCl2 and
5 mg GSTBcrAK, after a 10-min incubation at
37 8C; 3, 1 mm adenosine, 0.2 mm [g-32P]ATP,
5 mm MgCl2. Arrows indicate the positions
corresponding to radiolabelled AMP and ATP.
(B) Km and Vmax determinations of B. canis rossi
recombinant adenosine kinase.

containing the recombinant pGEX 4T-3/BcrAK overexpressed,


after IPTG induction, the GSTBcrAK which formed a major
band on SDS/PAGE of about 60 kDa, consistent with an insert
of cDNA encoding a 40-kDa protein fused to the GST (Fig. 2,
lane 2). The 42 8C growing phase of bacteria was used to
increase the amount of soluble GSTBcrAK in the culture,
improving the yield of purified enzyme [42]. After elution, this
purified GSTBcrAK was visualized by using a Coomassie
brilliant blue-stained polyacrylamide gel (Fig. 2, lane 3).
Enzymatic assays were carried out to confirm adenosine
kinase activity, according to our predictive model. The
adenosine kinase activity of the GSTBcrAK protein expressed
in and purified from E. coli was monitored by TLC after
incubation with [g-32P]ATP and adenosine, in the presence of
magnesium (Fig. 3A). A specific appearance of radiolabelled
AMP was evidenced with the recombinant GSTBcrAK
protein (Fig. 3A, lane 2), indicating that adenosine was phosphorylated by the BcrAK moiety. As predicted by analysis of
our model, no specific activity was revealed by TLC neither on
ribose 6-phosphate or fructose 6-phosphate nor using the GST
as control (data not shown). As expected, a low phosphorylation
rate (. 100-fold less than for adenosine) was observed when
the fructose was used as substrate (data not shown). However,
the kinetic experiment revealed that the BcrAK moiety of the
recombinant protein had an apparent Km for adenosine of 70 nm
and an apparent Vmax of 4.65  1023 mmolmin21 (Fig. 3B).
This Km value is in agreement with those obtained for the native
human heart adenosine kinase (57 nm), the purified human
Molt-4 T lymphoblast and the pig liver enzymes (75 and
46 nm, respectively) [12]. Thus, these studies strongly support
the predictive model of BcrAK and indicate that the cDNA
encodes an adenosine kinase rather than a sugar kinase. The full
coding region of BcrAK has been identified and this enzyme is
catalytically equivalent to the mammalian ones.
Since the preparation of this paper, the theoretical BcrAK
model has been compared with the recently solved crystal
structure of the related human adenosine kinase [43], confirming that these adenosine kinases adopt a fold similar to that
of EcRK despite the fact that human adenosine kinase is
monomeric. It also validates the predicted role of the
methionine M43 in the substrate specificity.

CONCLUSIONS
The cloning, sequencing and expression of an immunogenic
antigen of the B. canis rossi parasite is presented here. The
molecule corresponds to a new adenosine kinase belonging to

1020 C. Carret et al. (Eur. J. Biochem. 265)

the PFK-B family. This is the first characterization of a Babesia


canis enzyme as well as the first molecular characterization of
an adenosine kinase from an apicomplexan parasite. The
presence of a possible cleavable signal peptide in the BcrAK
sequence is an original feature. Indeed, this is the first
description of a putative secreted adenosine kinase and, even
if Babesia parasites release exo-antigens in their host's plasma
[37], it is surprising to find a metabolic enzyme excreted. The
molecular modelling, based on the crystal structure of EcRK,
suggests that BcrAK displays many specific characteristics.
The fine structure/function analysis of BcrAK indicates that the
residues involved in the ATP-binding site are strictly conserved
and cluster in three motifs in the C-terminus of the molecule. In
contrast, analysis of the active site suggests the presence of two
specific substitutions (H41 and M43) which are putatively
related to the substrate-binding recognition site for adenosine
compounds. The recombinant BcrAK Km and Vmax values
determined for the adenosine strongly validate the model. This
comparative analysis indicates the importance of studying
distantly related proteins at the structural level, especially to
direct research toward the right substrate. Solving the threedimensional structure should enhance our understanding of the
role of adenosine kinase and help in the development of
adenosine kinase inhibitors.

q FEBS 1999

11.

12.

13.

14.

ACKNOWLEDGEMENTS

15.

The authors would like to thank S. Camillieri and V. Arnavielle for efficient
technical assistance and C. Rouveirol for secretarial assistance. The authors
are also grateful to J. P. Pin and A. M. Cathiard for valuable help. We thank
H. Vial for critical reading of the manuscript. This work was supported by a
grant from Intervet International (the Netherlands). C. C. is a PhD student
supported by a fellowship from the Ministere de l'Education Nationale, de
la Recherche et de la Technologie and Internet International BV
(Convention Cifre no. 679/95).

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