Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI

The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg2+, the essential divalent metal ion cofactor which can be replaced by Mn2+ in vitro. We have carried out a mutational analysis of these presumptive Mg2+ ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg2+, demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg2+ but active in the presence of the thiophilic Mn2+ suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn2+ the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg2+ ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg2+ binding sites of PI-SceI is proposed.