Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif.
The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5' RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources.
Sinha
K M
KM
The Leishmania Group, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta-700032, India.
Ghosh
M
M
Das
I
I
Datta
A K
AK
D020106:Acrylamide / Q000378:metabolism; D000248:Adenosine Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D011193:Potassium Iodide / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020040:Trans-Splicing / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D011193:Potassium Iodide; D020106:Acrylamide; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000248:Adenosine Kinase