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The Thermus aualcus DNA methyltransferase M'Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of -CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethlonlne shows a/P folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide deft suitable to accommodate the DNA substrate. The N-terminal domain Is dominated by a nine-stranded 3-sheet; it contains the two conserved segments typical for N-methyltranserases which form a pocket for cofactor binding. The C-terminal domain is formed by four small 1-sheets and a-helices. The three-dimensional folding of M'Taq I is similar to that of the cytosine-speciflc Hha I methyltransferase, where the large 1-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.DNA-methyltransferases (MTases) are a family of enzymes that occur in nearly all living organisms. They catalyze the transfer of-CH3 from the cofactor S-adenosyl-L-methionine (AdoMet) to cytosine C5 (C-MTases) or cytosine N4 or adenine N6 (N-MTases) in di-to octanucleotide target sequences of double-stranded DNA (1). In bacteria, all three types of MTases are found and implicated in the protection of DNA from their own restriction endonucleases and in mismatch repair (2). In eukaryotes only C-MTases have been observed so far; they are involved in cell differentiation, genome imprinting, mutagenesis, and regulation of gene expression (3).The C-MTases are a homogeneous class of molecules with three-dimensional structures probably similar to the structure described recently for the M-Hha I enzyme from Haemophilus haemolyticus (4). This is because their amino acid sequences show sequential arrangement of 10 conserved segments (I to X) from the N to the C terminus (5); segments I (DXFXGXG, with X = any amino acid) and IV (FPCQ) are implicated in binding of AdoMet, and the cysteine in IV is involved in the transfer of -CH3. In contrast, the N-MTases show only two of the conserved segments (6). They correspond to segments I and IV in the C-MTases, namely I (DXFXGXG), which can degenerate so much that only one glycine is retained, and II (DPPY), where aspartate can be replaced by asparagine or seine, and tyrosine by phenylalanine. Because these two segments can occur in reversed order-i.e., one or the other N-terminal (7)-the N-MTases are a more heterogeneous class of molecules. When the amino acid sequences of only those N-MTases that recognize TNNA (N = any nucleotide) are compared, an additional segment III is found (8). It spans 38 amino acids, has no equivalent in C-MTases, and occurs sequentially-i.e., I, II, III. The mechanism of methyl transfer is different in C-and N-MTases. In the former the conserved cysteine SH in segment IV attacks C6 of cytosine to form a covalent intermediate with resonance-stabilized carbanionic ...

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