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 the gamma-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 approximately 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 AKgene 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 microM, respectively. The maximum enzyme activity (Vmax) was 0.18 micromol.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 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.
Villa
Héctor
H
Departamento de Farmacología y Toxicología (INTOXCAL), Facultad de Veterinaria, Universidad de León, Spain.
Pérez-Pertejo
Yolanda
Y
García-Estrada
Carlos
C
Reguera
Rosa M
RM
Requena
José María
JM
Tekwani
Babu L
BL
Balaña-Fouce
Rafael
R
Ordóñez
David
D
D000263:Adenylate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D000263:Adenylate Kinase; C110938:adenylate kinase 2