The Key Role of E418 Carboxyl Group in the Formation of Nt.BspD6I Nickase Active Site: Structural and Functional Properties of Nt.BspD6I E418A Mutant.

The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.