Identification and mutational analysis of Mg2+ binding site in EcoP15I DNA methyltransferase: involvement in target base eversion.
EcoP15I DNA methyltransferase catalyzes the transfer of the methyl group of S-adenosyl-l-methionine to the N6 position of the second adenine within the double-stranded DNA sequence 5'-CAGCAG-3'. To achieve catalysis, the enzyme requires a magnesium ion. Binding of magnesium to the enzyme induces significant conformational changes as monitored by circular dichroism spectroscopy. EcoP15I DNA methyltransferase was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 8.0. The inactivated enzyme was cleaved into two fragments with molecular masses of 36 and 35 kDa. Using this affinity cleavage assay, we have located the magnesium binding-like motif to amino acids 355-377 of EcoP15I DNA methyltransferase. Sequence homology comparisons between EcoP15I DNA methyltransferase and other restriction endonucleases allowed us to identify a PD(X)n(D/E)XK-like sequence as the putative magnesium ion binding site. Point mutations generated in this region were analyzed for their role in methyltransferase activity, metal coordination, and substrate binding. Although the mutant methyltransferases bind DNA and S-adenosyl-l-methionine as well as the wild-type enzyme does, they are inactive primarily because of their inability to flip the target base. Collectively, these data are consistent with the fact that acidic amino acid residues of the region 355-377 in EcoP15I DNA methyltransferase are important for the critical positioning of magnesium ions for catalysis. This is the first example of metal-dependent function of a DNA methyltransferase. These findings provide impetus for exploring the role(s) of metal ions in the structure and function of DNA methyltransferases.
Bist
Pradeep
P
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Rao
Desirazu N
DN
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D005296:Ferrous Compounds / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism* / Q000502:physiology; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D024342:Amino Acids, Acidic; D005296:Ferrous Compounds; C020748:ferrous sulfate; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D008274:Magnesium