Open AccessMolecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani
Author(s) -
Villa Héctor,
PérezPertejo Yolanda,
GarcíaEstrada Carlos,
Reguera Rosa M.,
Requena José María,
Tekwani Babu L.,
BalañaFouce Rafael,
Ordóñez David
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03826.x
Subject(s) - leishmania donovani , adenylate kinase , gene , leishmania , characterization (materials science) , biology , chemistry , computational biology , biochemistry , genetics , leishmaniasis , nanotechnology , materials science , enzyme , computer science , visceral leishmaniasis , parasite hosting , world wide web
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 γ‐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 genomic library of Leishmania 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 K m values of 104 and 74 µ m , respectively. The maximum enzyme activity ( V max ) was 0.18 µmol·min −1 · mg −1 protein. P 1 ,P 5 ‐(bis adenosine)‐5′‐pentaphosphate (Ap 5 A), the specific inhibitor of AK, competitively inhibited activity of the recombinant enzymes with estimated K i values of 190 n m and 160 n m for ATP and AMP, respectively. Ap 5 A also inhibited the growth of L. donovani promastigotes 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.