Mutational studies reveal lysine 352 on the large subunit is indispensable for catalytic activity of bi-subunit topoisomerase I from Leishmania donovani.
From the vanadate complex crystal structure of Leishmania donovani topoisomerase I, several amino acid residues have been implicated to be involved in the catalytic reaction. Although several predictions and propositions have been made, the exact role of these amino acids has not yet been clearly demonstrated in vitro. Among these residues, lysine 352 and arginine 314 stand as potential candidates for playing the role of a general acid during the cleavage step. In this study, we have characterized the role of lysine 352 on the large subunit, by site-directed mutagenesis and have tried to identify the general acid that can protonate the 5?-O atom of the leaving strand. Studies with the mutant enzymes reveal that, relaxation activity was severely affected when Lys352 was mutated to arginine or alanine (K352R or K352A). Mutation of Arg314 to Lys (R314K) has very little effect on the relaxation activity. Detailed study reveals that, both cleavage and religation steps are severely affected in case of K352R and K352A and the cleavage religation equilibrium is shifted towards the cleavage. On the contrary, the R314K mutant exhibits only a slightly slower rate of cleavage compared to wild-type enzyme. Cleavage assays with an oligonucleotide containing 5?-bridging phosphorothiolate indicate that Lys352 acts as a general acid in the cleavage step. Altogether, this study establishes the indispensable role of lysine 352 in the catalytic reaction of L. donovani topoisomerase I.
Ganguly
Agneyo
A
Indian Institute of Chemical Biology, Jadavpur, Kolkata, 700032, India.
Sengupta
Souvik
S
Bosedasgupta
Somdeb
S
Roy
Amit
A
Majumder
Hemanta K
HK
D000818:Animals; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics*; D008026:Ligation; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*
D004247:DNA; D004264:DNA Topoisomerases, Type I; D008239:Lysine