Leishmania donovani bisubunit topoisomerase I gene fusion leads to an active enzyme with conserved type IB enzyme function.
All eukaryotic topoisomerase I enzymes are monomeric enzymes, whereas the kinetoplastid family (Trypanosoma and Leishmania) possess an unusual bisubunit topoisomerase I. To determine what happens to the enzyme architecture and catalytic property if the two subunits are fused, and to explore the functional relationship between the two subunits, we describe here in vitro gene fusion of Leishmania bisubunit topoisomerase I into a single ORF encoding a new monomeric topoisomerase I (LdTOPIL-fus-S). It was found that LdTOPIL-fus-S is active. Gene fusion leads to a significant modulation of in vitro topoisomerase I activity compared to the wild-type heterodimeric enzyme (LdTOPILS). Interestingly, an N-terminal truncation mutant (1-210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-Delta(1-210)S], showed reduced topoisomerase I activity and camptothecin sensitivity in comparison to LdTOPIL-fus-S. Investigation of the reduction in enzyme activity indicated that the nonconserved 1-210 residues of LdTOPIS probably act as a 'pseudolinker' domain between the core and catalytic domain of the fused Leishmania enzyme, whereas mutational analysis of conserved His453 in the core DNA-binding domain (LdTOPIL) strongly suggested that its role is to stabilize the enzyme-DNA transition state through hydrogen bonding to one of the nonbridging oxygens. Taken together, our findings provide an insight into the details of the unusual structure of bisubunit topoisomerase I of Leishmania donovani.
Das
Benu B
BB
Department of Molecular Parasitology, Indian Institute of Chemical Biology, Kolkata, India.
Bose Dasgupta
Somdeb
S
Ganguly
Agneyo
A
Mazumder
Saumyabrata
S
Roy
Amit
A
Majumder
Hemanta K
HK
D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007893:Leishmania donovani / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004264:DNA Topoisomerases, Type I