title: Characterization of the DNA-binding domain and identification of the active site residue in the 'Gyr A' half of Leishmania donovani topoisomerase II.
abstract: DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. To investigate the biochemical properties of the individual domains of Leishmania donovani topoisomerase II, four truncation mutants were generated. Deletion of 178 aminoacids from the C-terminus (core and LdDeltaC1058) had no apparent effect on the DNA-binding or cleavage activities of the enzymes. However, when 429 aminoacids from the N-terminus and 451 aminoacids from the C-terminus were removed (LdDeltaNDeltaC), the enzyme was no longer active. Moreover, the removal of 429 aminoacids from the N-terminus (LdDeltaNDeltaC, core and LdDeltaN429) render the mutant proteins incapable of performing ATP hydrolysis. The mutant proteins show cleavage activities at wide range of KCl concentrations (25-350 mM). In addition, the mutant proteins, excepting LdDeltaNDeltaC, can also act on kDNA and linearize the minicircles. Surprisingly, the mutant proteins fail to show the formation of the enhanced cleavable complex in the presence of etoposide. Our findings suggest that the conformation required for interaction with the drug is absent in the mutant proteins. Here, we have also identified Tyr(775) through direct sequencing of the DNA linked peptide as the catalytic residue implicated in DNA-breakage and rejoining. Taken together, our results demonstrate that topoisomerase II are functionally and mechanistically conserved enzymes and the variations in activity seem to reflect functional optimization for its physiological role during parasite genome replication.
authors.affiliation: Molecular Parasitology Laboratory, Indian Institute of Chemical Biology 4 Raja S.C. Mullick Road, Kolkata-700032, India.
authors.forename: Tanushri
authors.initials: T
authors.lastname: Sengupta
authors.forename: Mandira
authors.initials: M
authors.lastname: Mukherjee
authors.forename: Rakhee
authors.initials: R
authors.lastname: Das
authors.forename: Aditi
authors.initials: A
authors.lastname: Das
authors.forename: Hemanta K
authors.initials: HK
authors.lastname: Majumder
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D007893:Leishmania donovani / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
chemical_list: D004268:DNA-Binding Proteins; D011189:Potassium Chloride; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II