'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani.
The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, 'LeishMan' topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.
Ganguly
Agneyo
A
Department of Molecular Parasitology, Indian Institute of Chemical Biology, 4 Raja S.C.Mullick Road, Jadavpur, Kolkata 700032, India.
Das
Benu Brata
BB
Sen
Nilkantha
N
Roy
Amit
A
Dasgupta
Somdeb Bose
SB
Majumder
Hemanta K
HK
D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D007893:Leishmania donovani / Q000201:enzymology*; D011188:Potassium / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000502:physiology; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D021122:Protein Subunits; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I; D011188:Potassium; D002166:Camptothecin