Molecular and functional characterization of adenylate kinase 2
gene from
Leishmania donovani
He
´
ctor Villa
1
, Yolanda Pe
´
rez-Pertejo
1
, Carlos Garcı
´
a-Estrada
1
, Rosa M. Reguera
1
, Jose
´
Marı
´
a Requena
2
,
Babu L. Tekwani
3
, Rafael Balan
˜
a-Fouce
1
and David Ordo

AK2 from a genomiclibrary of Leishmania donovani and also
its expression in leishmania promastigote cultures. AK2 was
localized on an 1.9-Mb chromosomal bandas a single copy
gene. L. donovani AK2 gene is expressed as a single 1.9-kb
mRNA transcript that is developmentally regulated and
accumulated during the early log phase. The overexpression
of L. donovani AK gene in Escherichia coli yielded a 26-kDa
polypeptide that could be refolded to a functional protein
with AK activity. The recombinant protein was purified to
apparent homogeneity. Kinetic analysis of purified
L. donovani AK showed hyperbolic behaviour for both ATP
and AMP, with K
m
values of 104 and 74 l
M
, respectively.
The maximum enzyme activity (V
max
) was 0.18 lmolÆmin
)1
Æ
mg
)1
protein. P
1
,P
5
-(bis adenosine)-5¢-pentaphosphate
(Ap
5

plastida (that includes trypanosomes, leishmanias and other
pathogenic parasites) has not been studied in detail yet. As
in their mammalian hosts, AK in these parasites seems to be
distributed in several intracellular compartments. These
eukaryotic microorganisms have some characteristic sub-
cellular organelles, such as modified mitochondria called
ÔkinetoplastsÕ and several specific energy-producing micro-
bodies called ÔglycosomesÕ [5]. AK plays an important role in
the ATP-regenerating system required for eukaryotic ciliary
or flagellar movements and has been found to be associated
with Tetrahymena cilia [6], Paramecium caudatum [7] as well
as vertebrate spermatozoid flagella [8,9]. AK activity in
Leishmania promastigotes and Trypanosoma spp. has been
found to be associated with the membrane of glycosomes
[10,11]. A third form of AK located in the cytosol has been
proposed as a virulence factor in bacteria, as it is secreted
along with other ATP-related enzymes, contributing to
modulation of ATP levels during macrophage death [12,13].
Structural studies with AKs from different sources have
revealed the presence of three distinct domains: the rigid
CORE-domain and two smaller peripheral domains or
mobile parts, the NMP-binding site and the LID-domain
[4]. The NMP-binding site has many intermolecular
contacts with the nucleotide phosphoryl acceptor (NMP),
whereas the LID-domain prevents the hydrolysis of the
Mg-bound phosphoryl donor in the active site. The relative
movement of the NMP-binding site and LID-domain
Correspondence to D. Ordo
´
n

EMBL and GenBank Nucleotide Sequence Databases under the
accession number AF156853.
(Received 14 May 2003, revised 25 July 2003,
accepted 9 September 2003)
Eur. J. Biochem. 270, 4339–4347 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03826.x
induced by the substrates leads to two conformations called
ÔclosedÕ and ÔopenÕ states, which have been thoroughly
described by Schulz et al. [14]. Genes encoding AKs have
been characterized in many organisms, including bacteria,
fungi and mammals. There are no reports about character-
ization of this enzyme in parasitic protozoa. Leishmania
donovani is the aetiological agent for visceral leishmaniasis, a
devastating disease which is mostly endemic in Asian and
Mediterranean countries [15]. This paper describes the
cloning and functional characterization of an AK gene from
a genomic library of L. donovani as well as its expression
and molecular characterization.
Materials and methods
Materials
Plasmids pGEM-3Zf(+) and pQE30 were from Promega
and QIAGEN, respectively. [
32
P]dCTP[aP] (3000 CiÆ
mmol
)1
) was from DuPont-NEN. Restriction enzymes
and Taq DNA polymerase were from Boehringer Mann-
heim. Isopropyl thio-b-
D
-galactoside (IPTG), pyruvate

cells in 10 m
M
EDTA, 150 m
M
NaCl,
0.4% SDS, 50 mgÆmL
)1
proteinase K and incubated at
65 °C for 1 h followed by an overnight incubation at 37 °C.
The DNA was further purified by phenol/chloroform
extraction and ethanol precipitation. RNA was isolated
with RNAeasy kit (QIAGEN) according to manufacture’s
protocol.
Isolation of a
L. donovani
AK gene fragment by PCR
To generate a DNA probe for the isolation of the
L. donovani AK gene, PCR was carried out using degenerate
oligonucleotides based on conserved amino acid sequences
in human AK type 2A [16], Saccharomyces cerevisiae [17],
Trichomonas vaginalis [18], Oryza sativa [19] and Escherichia
coli [20] AK proteins. The sense primer (5¢-GACGG
TTTTCCGCGCAC-3¢) corresponding to the amino acid
residues DGFPRT and an antisense primer (5¢-ACCAGG
GGCTCGCCGGT-3¢) corresponding to amino acid resi-
dues TGEPLV were used for isolation of AK gene fragment
by PCR with L. donovani genomic DNA. The PCR product
was sequenced on both strands by an ALF automated
sequencer (Sistemas Geno
´

through secondary and tertiary screenings, until all the
plaques on the plate appeared positive. The DNA was
isolated from amplified phages by liquid culture method as
described by Sambrook et al. [21].
Subcloning and sequencing of
L. donovani AK
gene
The isolated bacteriophage DNA was digested with restric-
tion endonucleases, separated by electrophoresis on 0.7%
agarose gels, and transferred to positively charged nylon
membranes by the method of Southern [22]. Southern blots
were probed with the 215-bp PCR product under the same
conditions described above for the screening of the genomic
library. A single 1.6-kb EcoRI–SphI fragment that hybrid-
ized to the probe was ligated into pGEM-3Zf(+) and
transformed into E. coli DH5a. Large-scale plasmid pre-
paration of pGEM-3Zf(+) containing the 1.6-kb EcoRI–
SphI fragment was processed using QIAGEN columns, and
the insert was sequenced as above. Analyses of nucleotide
and amino acid sequences were performed using the
BLAST
algorithm from the database National Center for Biotech-
nology Information.
Southern and Northern analyses
Genomic DNA prepared from L. donovani promastigotes
harvested at late log phase was digested with restriction
enzymes, as described above, separated by electrophoresis in
0.8% agarose gels and transferred to nylon membranes.
Total RNA was extracted from L. donovani promastigotes
using RNeasy Mini kit (QIAGEN), following the manu-

i
mixed
1 : 1 with 2% agarose. Processing of the samples was
carried out at 50 °Cfor48 hin10 mL0.5
M
EDTA pH 8.0,
1% Sarkosyl, and 150 lLof2mgÆmL
)1
freshly prepared
proteinase K. Separation of the chromosomal bands was
achieved by electrophoresis at 14 °Cin1%agarosegels
with 0.5 · TBE running buffer, using a Clamped Homo-
geneous Electrical Field apparatus (CHEF, BIO-RAD)
with a 35–120 s ramping pulse at 6 VÆcm
)1
for 33 h.
S. cerevisiae (Amersham) chromosomes were used as
molecular weight markers. After staining with ethidium
bromide, gels were blotted onto nylon filters (Sigma) by
alkaline transfer. The membrane was probed to the 690-bp
probe as described above.
Heterologous expression
The AK gene was amplified by PCR from L. donovani
genomic DNA. The sense primer 5¢-ACATGCATGCAT
GAAGATCGTGATGGAAGG-3¢ introduces an SphI
restriction site and the antisense one 5¢-AACTGCAG
GCTTTCACCAGAATTTCCACC-3¢ introduces a PstI
restriction site to subclone the AK gene in the pGEM-
3Zf(+) cloning vector (Promega), creating pGEM-AK. To
subclone the AK gene into pQE-30 expression vector

urea,
20% Triton-X100, 50 m
M
Tris/HCl pH 8, 10 m
M
MgCl
2
,
1m
M
dithiothreitol and twice with 50 m
M
Tris/HCl (pH 8),
10 m
M
MgCl
2
,1m
M
dithiothreitol. After incubation with
50 lgÆmL
)1
DNAse I (Roche), cell debris were removed by
centrifugation and the insoluble fraction was dissolved in
equilibration buffer (10 m
M
Tris/HCl, 300 m
M
NaCl con-
taining 8

urea overnight [23].
SDS/PAGE and Western blotting
L. donovani promastigotes were harvested during exponen-
tial growth phase (day 3) and washed twice with NaCl/P
i
.
Once they were sonicated and centrifuged at 10 000 g for
20 min, the supernatant was removed. Fifty lg protein were
diluted in loading buffer (60 m
M
Tris/HCl pH 6.8, 2%
SDS, 5% 2-mercaptoethanol, 5% glycerol), heated in a
boiling water bath for 5 min, and analysed by SDS/PAGE
(12% acrylamide, 2.7% bisacrylamide). Proteins were
electrotransferred to nylon membranes for 1 h at
25–30 VÆcm
)1
, blots were blocked by incubation in 10 m
M
Tris/HCl pH 7.5, 1
M
NaCl, 0.5% Tween 20, 5% nonfat
milk powder (w/v) for 1 h at room temperature. Primary,
polyclonal antibodies (obtained from rabbit serum ino-
culated with purified recombinant leishmanial AK) were
added to this buffer and the blot incubated for an 2 h. The
blot was washed extensively in 10 m
M
Tris/HCl pH 7.5, 1
M

(range 0.010–1.5 m
M
). Protein content was estimated by
using the method of Bradford [25]. For analysis of enzyme
kinetics AK assay was done at varying concentrations of
ATP and AMP and the results were analysed by double
reciprocal Lineweaver–Burk plot by using
SIGMA PLOT
.
Kinetic parameters (K
m
and V
max
) were computed from
these plots.
In vitro
anti-leishmania assay
Anti-leishmanial activity of Ap
5
A the inhibitor of AK was
tested on a transgenic cell line of L. donovani promastigotes
expressing firefly luciferase. These cells show constant and
stable expression of luciferase, which is directly proportional
to the number of live promastigotes. The assay was
performed in clear-bottomed, 96-well micro plates. Promas-
tigote culture (200 lL; 2 · 10
6
cells per well) was exposed to
varying concentrations (5–250 l
M

amino acids, with an estimated molecular mass of
26 kDa. This gene was designated the AK gene (see
GenBank accession number AF156853). The amino acid
sequence deduced from this ORF showed significant
homology with AK enzymes from different organisms.
Multiple sequence alignment (Fig. 1) of the L. donovani
AK protein with other proteins from phylogenetically
diverse organisms revealed that the Leishmania AK gene
has the conserved motifs involved in NMP binding in a
sequence from 30 to 61 amino acids. The amino acids
residues that possibly mediate the interactions with AMP
are T31, R36 and L59. In position 35 a valine residue
replaced the leucine residue, which is conserved in all
other sequences, while at position 60 an isoleucine residue
was present in place of a highly conserved valine. The
decapeptide GPPQGGKTTV (in position 7–16) in
L. donovani AK resembles the P-loop related to the ATP-
binding site [26]. The sequence containing four cysteine
residues C-X
2
-C-X
n
-C-X
2
-C predicted to be a zinc-
binding motif was not found in L. donovani LID
sequence, although it was present in P. falciparum AK
(AF308612), where it is located in positions 127–158. The
LID domain, said to be related to the active site of the
enzyme in most AK, starts at position 124 and extends to

higher during early period (days 1 and 2) of growth,
Fig. 1. Multiple amino acid sequence align-
ments of L. donovani AK. The predicted amino
acid sequences for L. donovani, Trypanosoma
brucei, T. vaginalis, P. falciparum, S. cerevisi-
ae, and human 2A AKs were aligned using the
CLUSTAL X
multiple sequence alignment pro-
gram. Symbols: Ô*Õ identical or conserved
residues in all sequences in the alignment; Ô:Õ
conserved substitutions; Ô.Õ semiconserved
substitutions. Complete genomic sequences
of AK proteins are available from the
GenBank, for L. donovani accession number
(AF156853), T. brucei (AF047722),
T. vaginalis (U07203), P. falciparum
(AF308612), S. cerevisiae (Y00413) and
human 2A (U39945).
4342 H. Villa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
diminishing to very low levels in the logarithmic and
stationary phases (Fig. 3C).
Overexpression and purification of AK2
The cultures of bacterial cells (XL1-Blue) transformed with
pQE30-AK were induced with IPTG for overexpression of
AK2 gene. Marked overexpression of AK2 protein was
noticed but the recombinant AK protein accumulated
mainly in the inclusion bodies (Fig. 4A). A 26-kDa
polypeptide was detected after induction of the culture with
IPTG, which matched the molecular mass predicted from
the amino acid sequence of AK2 protein. The purification

not shown). Maximum enzyme activity (V
max
)atsteady-
state conditions and saturation concentrations of both
substrates in the forward reaction, i.e. 2.5 m
M
ATP and
1.5 m
M
AMP, was 0.18 lmol ADP formedÆmin
)1
Æmg
)1
.
The enzyme exhibited hyperbolic behaviour with both
ATP and AMP. The K
m
value estimated for ATP was
104 ± 20 l
M
, while for AMP it was 74 ± 18 l
M
.
Stability of the recombinant leishmania AK was also
checked. The recombinant enzyme protein was incubated,
as described previously, at 4 °C, 13 °Cand26.5°Cfor10
consecutive days. Activity was measured at different time
Fig. 2. Southern blot analysis of wild-type AK loci. (A) Genomic DNA
(20 lg) was isolated from L. donovani promastigotes, digested with
PvuI, NotI, SalI, KpnI, BamHI, PstI, and resolved on a 0.7% agarose

5
A, was studied
on L. donovani recombinant AK under standard assay
conditions (Fig. 6). Different concentrations (50–1000 n
M
)
of Ap5A were added to the assay buffer, which contained
6 lg refolded AK and three concentrations of one of the
nucleotides (ATP: 0.62–2.5 m
M
; AMP: 0.5–1.5 m
M
) under
forward assay conditions (Fig. 6A,B). Dixon analyses of
the inhibition of leishmania AK with Ap
5
A at saturating
concentrations of the other substrate (ATP: 2.5 m
M
;AMP
1.5 m
M
), showed a competitive inhibitory pattern for both
substrates, with K
i
values of 190 n
M
and 160 n
M
for ATP

ble fraction; lane 2 insoluble fractions. Lane 3 shows the inclusion
bodies after purification as outlined in Materials and methods. The
arrow shows the 26-kDa expression product. (B) Forty micrograms
exponential phase (day 3) promastigotes were loaded onto SDS/12%
PAGE. The separated proteins were transblotted onto nylon mem-
branes and probed with an anti-AK polyclonal antibody. MWM,
molecular mass markers; CNT, preimmune serum.
Fig. 5. Half-life of recombinant L. donovani AK at different tempera-
tures. Inclusion bodies from E. coli XL1-Blue, transformed with PQE-
30-AK, were washed, folded and purified as described in Materials and
methods. Purified enzyme aliquots were maintained at 4 °C(m), 13 °C
(j) and 26.5 °C(d) and AK activity was measured at different time
points. Each point represents the mean of two different experiments.
Fig. 6. Dixon plots of the inhibition of recombinant AK by the specific
inhibitor Ap
5
A. Six micrograms recombinant L. donovani AK were
incubated in presence of several concentrations of ATP (A) or AMP
(B) and different concentrations of the inhibitor. Each point is the
mean of three separate trials.
4344 H. Villa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Discussion
Amino acid sequence of AK2 cloned from L. donovani
shows significant homology with the corresponding
enzyme from S. cerevisiae (28%) [17], Homo sapiens
(29%) [16], P. falciparum (15%) (AF308612), T. vaginalis
(25%) [18] and T. brucei rhodesiense (14%) (AF047722).
Analysis of leishmania AK sequence by
BLAST
and

as a metal cofactor. Three different
views of a theoretical three-dimensional ribbon model
diagram of the leishmania AK2, which were obtained
using the
SWISS-MODEL
(Automated-Knowledge-Based-
Protein-Modelling-Server; ) (Fig. 8),
shows the structure of leishmania AK2 to be similar to
Fig. 7. Effect of Ap5A on the growth of L. donovani promastigotes
in vitro. Effect of Ap5A at varying concentrations (5–250 l
M
)was
tested alone (control) and also in combination with ATP or ADP
(1 m
M
). Each point represents the mean ± SD of at least triplicate
observations.
Fig. 8. Three-dimensional ribbon diagram of L. donovani AK 2. The
SWISS-MODEL
(Automated-Knowledge-Based-Protein-Modelling-Ser-
ver; ) was used for prediction of theoretical three-
dimensional structure of the enzyme. The three different views were
obtained by simple rotation of the same model. The flexible LID
domain appears at top of the three figures, NMP-binding site and ATP
binding domain are displayed, NH
2
- and COOH-termini are also
indicated.
Ó FEBS 2003 L. donovani adenylate kinase (Eur. J. Biochem. 270) 4345
that of E. coli AK. The flexible LID domain which

logy in nucleotide sequence was found to be 94%. Other
putative AK genes were significantly different. The results of
Southern blotting also indicated the AK2 as a single copy
gene in L. donovani. Investigation of transcription of the AK
gene by Northern blot analysis during the Leishmania life
cycle detects a single mRNA transcript of  1.9 kb.
Leishmania cell seem to have strong regulation for expres-
sion of AK2 gene as the relative abundance of AK mRNA
during the early logarithmic phase was much higher than in
other phases of the promastigote cell cycle. AK has been
detected in glycosomes in Leishmania [10]. Recently, three
new putative AK gene sequences, one of them is incomplete
(AL 139794), have been identified by the L. major Friedlin
Genome Project. One of the L. major AK genes seems to
encode for a short AK isoform (AQ 852692), with an LID
domain of 18 amino acids, while the other two genes
(AL354533) encode longer AKs. Moreover, the AL354533
and AQ 852692 isoforms lack the threonine 31 residue,
which is replaced by a valine or a serine, respectively. This
suggests possible involvement of AKs also in CMP/UMP
metabolism in leishmania. The recombinant leishmanial
AK2wasalsoselectivelyrecognizedbytheserumof
hamsters infected with L. donovani (data not shown). AK2
may be a potential target antigen for a vaccine or diagnosis
of leishmaniasis. The sequence of the AK genes identified in
the leishmania genome, including the present one, differ
significantly from each other. Diverse AK isoforms may be
localized in different cellular compartments in leishmania
and may have important roles in energy metabolism and
adenine nucleotide equilibrium. Inhibition of leishmania

rocytes. AK may also play a role in the ATP regeneration
system required for flagellar movements in leishmania
promastigotes as shown earlier in the cilia of Paramecium
caudatum [7] or may also be a virulence factor as shown in
Pseudomonas [12]. AK may thus be a potential drug target
in leishmania.
Acknowledgements
This work was supported in part by Comisio
´
n Interministerial
de Ciencia y Tecnologı
´
a (CICYT, grants PM98/0036 and
BMC2002 04107-C02-02) and Junta de Castilla y Leo
´
n (JCyL grants
LE01/00B and LE54/03). We also thank to Dr J. Zhou for his advice in
AK refolding experiments, Dr N. Fasel, I. Segura, P. Bastien, and
F. Fierro for their technical support in PFGE. BLT is supported by
Cooperative scientific agreement grant from Center for Disease
Control, Atlanta USA (U50-CCU418839) and also by a USDA
cooperative agreement no. 58-6408-2-0009.
References
1. Noda, L.H. (1973) Adenylate kinase. In The Enzymes,3rdedn,
Vol. 8, (Boyer, P.D., ed.), pp. 279–305. Academic Press, New
York.
2. Yan, H. & Tsai, M.D. (1999) Nucleoside monophosphate kinases:
structure, mechanism, and substrate specificity. Adv. Enzymol.
Relat. Areas Mol. Biol. 73, 103–134.
3. Bandlow, W., Strobel, G. & Schricker, R. (1998) Influence of

cultured procyclic trypomastigotes of Trypanosoma. Mol. Bio-
chem. Parasitol. 4, 291–309.
12. Markaryan, A., Zaborina, O., Punj, V. & Chakrabarty, A.M.
(2001) Adenylate kinase as a virulence factor of Pseudomonas
aeruginosa. J. Bacteriol. 183, 3345–3352.
13. Punj, V., Zaborina, O., Dhiman, N., Falzari, K., Bagdasarian, M.
& Chakrabarty, A.M. (2000) Phagocytic cell killing mediated by
secreted cytotoxic factors of Vibrio cholerae. Infect. Immun. 684,
930–4937.
14. Schulz, G.E. (1992) The induced-fit movements in adenylate
kinases. Faraday Discuss. 93, 85–93.
15. Balan
˜
a-Fouce,R.,Reguera,R.M.,Cubrı
´
a, J.C. & Ordo
´
n
˜
ez, D.
(1998) The pharmacology of leishmaniasis. Gen. Pharmacol. 30,
435–443.
16. Lee,Y.,Kim,J.W.,Lee,I.A.,Kang,H.B.,Choe,Y.K.,Lee,H.G.
Lim, J.S., Kim, H.J., Park, C. & Choe, I.S., (1996) Cloning and
characterization of cDNA for human adenylate kinase 2 A.
Biochem. Mol. Biol. Int. 39, 833–842.
17. Proba, K., Tomasselli, A.G., Nielsen, P. & Schulz, G.E. (1987)
The cDNA sequence encoding cytosolic adenylate kinase from
baker’s yeast (Saccharomyces cerevisiae). Nucleic Acids Res. 15,
7187.

Nakazawa, A. (2001) Cloning and characterization of adenylate
kinase from Chlamydia pneumoniae. J. Biol. Chem. 276,
13490–13498.
28. Berry, M.B. & Phillips, G.N. Jr, (1998) Crystal structures of
Bacillus stearothermophilus adenylate kinase with bound Ap5A,
Mg
2+
Ap5A, and Mn
2+
Ap5A reveal an intermediate lid position
and six coordinate octahedral geometry for bound Mg
2+
and
Mn
2+
. Proteins 32, 276–288.
29. Kanaani, J. & Ginsberg, H. (1989) Metabolic interconnection
between human malaria parasite Plasmodium falciprum and its
host erythrocyte: regulation of ATP levels by means of an
adenylate translocator and adenylate kinase. J. Biol. Chem. 264,
3194–3199.
Ó FEBS 2003 L. donovani adenylate kinase (Eur. J. Biochem. 270) 4347