Biochemical Characterization of the Topoisomerase Domain of M. kandleri Topoisomerase V [Enzymology]

August 18th, 2014 by Rajan, R., Osterman, A. K., Gast, A. T., Mondragon, A.

Topoisomerases are ubiquitous enzymes that modify the topological state of DNA inside the cell and are essential for several cellular processes. Topoisomerase V (Topo-V) is the sole member of the type IC topoisomerase subtype. The topoisomerase domain has a unique fold amongst topoisomerases and the putative active site residues show a distinct arrangement. The present study was aimed at identifying the roles of the putative active site residues in the DNA cleavage/religation process. Residues R131, R144, H200, E215, K218, and Y226 were mutated individually to a series of conservative and non-conservative amino acids and the DNA relaxation activity at different pHs, times, and enzyme concentrations was compared to wild-type activity. The results suggest that R144 is essential for protein stability since any substitution at this position was deleterious, and that R131 and H200 are involved in transition state stabilization. E215 reduces the DNA binding ability of Topo-V, especially in shorter fragments with a fewer number of helix-hairpin-helix DNA binding motifs. Finally, K218 appears to play a direct role in catalysis, but not in charge stabilization of the protein-DNA intermediate complex. The results suggest that even though catalytically important residues are oriented in different fashions in the active sites of type IB and type IC topoisomerases, similar amino acids play equivalent roles in both of these subtypes of enzymes, showing convergent evolution of catalytic mechanism.