title: ATP independent type IB topoisomerase of Leishmania donovani is stimulated by ATP: an insight into the functional mechanism.
abstract: Most type IB topoisomerases do not require ATP and Mg(2+) for activity. However, as shown previously for vaccinia topoisomerase I, we demonstrate that ATP stimulates the relaxation activity of the unusual heterodimeric type IB topoisomerase from Leishmania donovani (LdTOP1L/S) in the absence of Mg(2+). The stimulation is independent of ATP hydrolysis but requires salt as a co-activator. ATP binds to LdTOP1L/S and increases its rate of strand rotation. Docking studies indicate that the amino acid residues His93, Tyr95, Arg188 and Arg190 of the large subunit may be involved in ATP binding. Site directed mutagenesis of these four residues individually to alanine and subsequent relaxation assays reveal that the R190A mutant topoisomerase is unable to exhibit ATP-mediated stimulation in the absence of Mg(2+). However, the ATP-independent relaxation activities of all the four mutant enzymes remain unaffected. Additionally, we provide evidence that ATP binds LdTOP1L/S and modulates the activity of the otherwise ATP-independent enzyme. This study establishes ATP as an activator of LdTOP1L/S in the absence of Mg(2+).
authors.affiliation: Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4 Raja S C Mullick Road, Kolkata 700032, India.
authors.forename: Souvik
authors.initials: S
authors.lastname: Sengupta
authors.forename: Agneyo
authors.initials: A
authors.lastname: Ganguly
authors.forename: Amit
authors.initials: A
authors.lastname: Roy
authors.forename: Somdeb
authors.initials: S
authors.lastname: Bosedasgupta
authors.forename: Ilda
authors.initials: I
authors.lastname: D'Annessa
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
authors.forename: Hemanta K
authors.initials: HK
authors.lastname: Majumder
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D011189:Potassium Chloride / Q000737:chemistry
chemical_list: C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D002413:Cations, Divalent; D004278:DNA, Superhelical; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D004264:DNA Topoisomerases, Type I; D008274:Magnesium