tailieunhanh - Báo cáo khoa học: Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani

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 thec-phosphate group from ATP to AMP, releasing twomolecules ofADP. This study describes cloningand functional characterizationof the gene encoding AK2fromagenomic libraryofLeishmaniadonovaniandalso its expression in leishmania promastigote cultures. | Eur. J. Biochem. 270 4339-4347 2003 FEBS 2003 doi Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani Hector Villa1 Yolanda Perez-Pertejo1 Carlos Garcia-Estrada1 Rosa M. Reguera1 Jose Maria Requena2 Babu L. Tekwani3 Rafael Balana-Fouce1 and David Ordonez1 1 Departamento de Farmacologia y Toxicologia INTOXCAL Facultad de Veterinaria Universidad de Leon Spain 2Centro de Biologia Molecular Severo Ochoa Universidad Autonoma de Madrid Spain 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 y-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 w chromosomal band as a single copy gene. L. donovani AK2 gene is expressed as a single 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 M respectively. The maximum enzyme activity Vmax was pmol-min-1-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 also inhibited the growth