A mutational analysis of the two motifs common to adenine methyltransferases.

All methyltransferases that use S‐adenosyl methionine as the methyl group donor contain a sequence similar to (D/E/S)XFXGXG which has been postulated to form part of the cofactor binding site. In N6‐adenine DNA methyltransferases there is a second motif, (D/N)PP(Y/F), which has been proposed to play a role similar to the catalytically essential PC motif conserved in all C5‐cytosine DNA methyltransferases. We have made a series of amino acid changes in these two motifs in the EcoKI N6‐adenine DNA methyltransferase. The mutant enzymes have been purified to homogeneity and characterized by physical biochemical methods. The first G is the most conserved residue in motif I. Changing this G to D completely abolished S‐adenosyl methionine binding, but left enzyme structure and DNA target recognition unaltered, thus documenting the S‐adenosyl methionine binding function of motif I in N6‐adenine methyltransferases. Substitution of the N with D, or F with either G or C, in motif II abolished enzyme activity, but left S‐adenosyl methionine and DNA binding unaltered. Changes of F to Y or W resulted in partial enzyme activity, implying that an aromatic residue is important for methylation. The substitution of W for F greatly enhanced UV‐induced cross‐linking between the enzyme and S‐adenosyl methionine, suggesting that the aromatic residue is close in space to the methyl‐group donor.