Three-dimensional structure of the adenine-specific DNA methyltransferase M.Taq I in complex with the cofactor S-adenosylmethionine.

Abstract

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 ...

Two Alternative Conformations of S-Adenosyl-L-homocysteine Bound to Escherichia coli DNA Adenine Methyltransferase and the Implication of Conformational Changes in Regulating the Catalytic Cycle

“…Such preferential interaction with AdoMet is in agreement with the preferred binding of AdoMet as compared with AdoHcy shown here. In agreement with these considerations, in all DNA MTases AdoMet adopts the extended conformation, and in the only examples where AdoMet and AdoHcy binding can be directly compared (M.TaqI and M.RsrI) AdoMet is extended whereas AdoHcy is folded (14,33). Therefore, the extended conformation most likely is the preferred conformation for AdoMet, whereas AdoHcy can adopt both the extended and the folded conformations in the binary complex.…”

Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

“…The highly conserved Asn in motif IV (Fig. 1) of the N-methyltransferase domains of peptide synthetases is also found in methyltransferases such as TaqI and glycine N-methyltransferase from rat (35,36,42). Cheng et al (13) have suggested that the side chain of Asn acts as a donor in a hydrogen bond to the target adenine and therefore allows direct transfer of the activated methyl group of AdoMet.…”

“…X-ray structure determination of different methyltransferases revealed a considerable flexibility concerning the location of motif I in the polypeptide chains. In the case of the TaqI and HhaI DNA methyltransferases, motif I is located near the N terminus, forming the AdoMet-binding site, whereas the DNAbinding site is C-terminal (13,(35)(36)(37). In contrast, in the case of the PvuII DNA methyltransferase, motif I is located closer to the C terminus (38).…”

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4‐dimethylamino‐l‐phenylalanine precursor of pristinamycin I

“…The product of ORF4 (named papM ) weakly resembled the TaqI methyltransferase from Thermophilus aquaticus (Barany et al, 1992) involved in the N-methylation of the adenine of the DNA recognition sequence. Careful sequence comparison with other S-adenosyl-L-methionine (SAM)-dependent methyltransferases revealed in PapM (amino acids 124 to 135) the conserved sequence rich in glycine residues (VLE/DXGXGXG) (Ingrosso et al, 1989) which forms part of the SAM-binding site in M-TaqI-AdoMet and M-HhaI-AdoMet crystalized complexes (Labahn et al, 1994;Cheng et al, 1993).…”