A MUTATIONAL ANALYSIS OF THE 2 MOTIFS COMMON TO ADENINE METHYLTRANSFERASES

All methyltransferases that use S-adenosyl methionine as the methyl gr oup donor contain a sequence similar to (D/E/S)XFXGXG which has been p ostulated 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 b een proposed to play a role similar to the catalytically essential PC motif conserved in all CS-cytosine DNA methyltransferases. We have mad e a series of amino acid changes in these two motifs in the EcoKI N6-a denine DNA methyltransferase. The mutant enzymes have been purified to homogeneity and characterized by physical biochemical methods. The fi rst G is the most conserved residue in motif I. Changing this G to D c ompletely abolished S-adenosyl methionine binding, but left enzyme str ucture and DNA target recognition unaltered, thus documenting the S-ad enosyl methionine binding function of motif I in N6-adenine methyltran sferases. Substitution of the N with D, or F with either G or C, in mo tif II abolished enzyme activity, but left S-adenosyl methionine and D NA binding unaltered. Changes of F to Y or W resulted in partial enzym e activity, implying that an aromatic residue is important for methyla tion. The substitution of W for F greatly enhanced UV-induced cross-li nking between the enzyme and S-adenosyl methionine, suggesting that th e aromatic residue is close in space to the methyl-group donor.