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 whi ch 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 Asp 326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal s tructure with the DNA substrate has been determined, suggests that Asp218 a nd Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center I I serve as ligands for Mg2+, the essential divalent metal ion cofactor whic h can be replaced by Mn2+ in vitro. We have carried out a mutational analys is of these presumptive Mg2+ ligands. The variants carrying an alanine or a sparagine substitution bind DNA, but (with the exception of the D229N varia nt) are inactive in DNA cleavage in the presence of Mg2+, demonstrating tha t these residues are important for cleavage. Our finding that the PI-SceI v ariants carrying single cysteine substitutions at these positions are inact ive 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 a re 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 enzym e, 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 infor mation, a model for the composition of the two Mg2+ binding sites of PI-Sce I is proposed.