Tài liệu Báo cáo khoa học: Molecular and functional characterization of adenylate kinase 2 gene from

Molecular and functional characterization of adenylate kinase 2

gene from Leishmania donovani

He´ ctor Villa1

, Yolanda Pe´rez-Pertejo1

, Carlos Garcı´a-Estrada1

, Rosa M. Reguera1

, Jose´ Marı´a Requena2

,

Babu L. Tekwani3

, Rafael Balan˜ a-Fouce1 and David Ordo´ n˜ ez1

1

Departamento de Farmacologı´a y Toxicologı´a (INTOXCAL), Facultad de Veterinaria, Universidad de Leo´n, Spain; 2

Centro de

Biologı´a Molecular ‘Severo Ochoa’, Universidad Auto´noma de Madrid, Spain; 3

National Center for Natural Products Research,

School of Pharmacy, University of Mississippi, USA

ATP-regenerating enzymes may have an important role in

maintaining ATP levels in mitochondria-like kinetoplast

organelle and glycosomes in parasitic protozoa. Adenylate

kinase (AK) (ATP:AMP phosphotransferase) catalyses the

reversible transfer of the c-phosphate group from ATP to

AMP, releasing two molecules of ADP. This study describes

cloning and functional characterization of the gene encoding

AK2 from a genomiclibrary ofLeishmania donovani and also

its expression in leishmania promastigote cultures. AK2 was

localized on an
1.9-Mb chromosomal band as 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 Km values of 104 and 74 lM, respectively.

The maximum enzyme activity (Vmax) was 0.18 lmolÆmin)1

Æ

mg)1 protein. P1,P5-(bis adenosine)-5¢-pentaphosphate

(Ap5A), the specific inhibitor of AK, competitively inhibited

activity of the recombinant enzymes with estimated Ki values

of 190 nM and 160 nM for ATP and AMP, respectively.

Ap5A alsoinhibited the growth ofL. donovanipromastigotes

in vitro which could be only partially reversed by the addition

of ADP. Thus, presence of a highly regulated AK2, which

may have role in maintenance of ADP/ATP levels in

L. donovani, has been demonstrated.

Keywords: adenylate kinase; functional expression; leish￾mania.

Adenylate kinase (AK) (ATP:AMP phosphotransferase,

EC 2.7.4.3) catalyses the reversible transfer of the c-phos￾phate group from ATP to AMP, releasing two molecules of

ADP [1]. Different isoenzymic forms of AK are involved in

maintenance of constant intracellular levels of adenine

nucleotides, necessary for energy metabolism and nucleic

acid synthesis [2]. Three AK isoforms have been described in

mammals: AK1 in the cytosol, AK2 in the mitochondrial

intermembrane space [3] and AK3, a GTP:AMP phospho￾transferase that resides exclusively in the mitochondrial

matrix [4].

The role of AKs in the organisms of the order Kineto￾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

kinetoplasts and several specific energy-producing micro￾bodies called glycosomes [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˜ez, Department Farmacologı´a y Toxi￾cologı´a (INTOXCAL), Ftad. Veterinaria, Universidad de Leo´n,

Campus de Vegazana s/n 24071 Leo´n, Spain.

Fax: + 34 987 291 252, Tel.: + 34 987 291 590,

E-mail: [email protected]

Abbreviations: AK, adenylate kinase; IPTG, isopropyl thio-b-D￾galactoside; Ap5A, P1,P5-bis(adenosine)-5¢-pentaphosphate; NMP,

nucleoside monophosphate; IC50, 50% inhibitory concentration.

Enzymes: adenylate kinase (ATP:AMP phosphotransferase;

EC 2.7.4.3).

Note: The nucleotide sequence data reported has been submitted to

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) FEBS 2003 doi:10.1046/j.1432-1033.2003.03826.x