Characterization of the ATPase activity of topoisomerase II from Leishmania donovani and identification of residues conferring resistance to etoposide.
We have cloned and expressed the 43 kDa N-terminal domain of Leishmania donovani topoisomerase II. This protein has an intrinsic ATPase activity and obeys Michaelis-Menten kinetics. Cross-linking studies indicate that the N-terminal domain exists as a dimer both in the presence and absence of nucleotides. Etoposide, an effective antitumour drug, traps eukaryotic DNA topoisomerase II in a covalent complex with DNA. In the present study, we report for the first time that etoposide inhibits the ATPase activity of the recombinant N-terminal domain of L. donovani topoisomerase II. We have modelled the structure of this 43 kDa protein and performed molecular docking analysis with the drug. Mutagenesis of critical amino acids in the vicinity of the ligand-binding pocket reveals less efficient inhibition of the ATPase activity of the enzyme by etoposide. Taken together, these results provide an insight for the development of newer therapeutic agents with specific selectivity.
Sengupta
Tanushri
T
Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, Kolkata-700032, India.
Mukherjee
Mandira
M
Das
Aditi
A
Mandal
Chhabinath
C
Das
Rakhee
R
Mukherjee
Tanmoy
T
Majumder
Hemanta K
HK
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004351:Drug Resistance; D005047:Etoposide / Q000494:pharmacology*; D006868:Hydrolysis; D007893:Leishmania donovani / Q000201:enzymology*; D047311:Luteolin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D059005:Topoisomerase II Inhibitors; D014315:Triterpenes / Q000494:pharmacology; D000094062:Betulinic Acid
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II; D005047:Etoposide; D047311:Luteolin; D059005:Topoisomerase II Inhibitors; D014315:Triterpenes; C098641:dihydrobetulinic acid; D000094062:Betulinic Acid