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.
Willcock
D F
DF
Institute of Cell and Molecular Biology, University of Edinburgh, UK.
Dryden
D T
DT
Murray
N E
NE
D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
D012436:S-Adenosylmethionine; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000225:Adenine