PMID:8070417
| Citation |
Willcock, DF, Dryden, DT and Murray, NE (1994) A mutational analysis of the two motifs common to adenine methyltransferases. EMBO J. 13:3902-8 |
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| Abstract |
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. |
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| Keywords |
Adenine/metabolism; Amino Acid Sequence; Base Sequence; DNA/metabolism; DNA Mutational Analysis; Escherichia coli/enzymology; Escherichia coli/genetics; Molecular Sequence Data; S-Adenosylmethionine/metabolism; Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics; Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism; Structure-Activity Relationship |
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