What is the catalytic mechanism of DNA (cytosine-5) methyltransferases? 
['All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position.', 'Kinetic and catalytic mechanism of HhaI methyltransferase.', 'Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI are described. With poly(dG-dC) as substrate, the reaction proceeds by an equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA.', 'Our studies reveal that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves attack of the C6 of substrate cytosines by an enzyme nucleophile and formation of a transient covalent adduct.', 'Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited: covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme.', 'We propose that correct positioning of the flipped base and the cofactor and binding to the transition state of methyl group transfer are the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl group transfer.', 'Co-transfections of reporter plasmids and plasmids encoding the catalytic domain of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene expression. As Dnmt3a mutants with C-->A and E-->A exchanges in the conserved PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, we checked for their catalytic activity in vitro.', 'In contrast, enzyme variants carrying E-->A, E-->D or E-->Q exchanges of the ENV glutamate are catalytically almost inactive, demonstrating that this residue has a central function in catalysis.', 'Therefore, covalent complex formation is not essential in the reaction mechanism of Dnmt3a. We propose that correct positioning of the flipped base and the cofactor and binding to the transition state of methyl group transfer are the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl group transfer.', 'Our results point to a possible connection between the catalytic mechanism of C5 Mtases and of enzymes that transfer methyl groups to N(4) of cytosine.', 'Previous X-ray crystallographic studies have revealed that the catalytic domain of a DNA methyltransferase (Mtase) generating C5-methylcytosine bears a striking structural similarity to that of a Mtase generating N6-methyladenine.', 'Spontaneous and reversible proton transfer between a conserved Glu in the active site and cytosine N3 at the transition state was observed in our simulations, revealing the chemical participation of this Glu residue in the catalytic mechanism.', 'The mechanism of DNA cytosine-5-methylation catalyzed by the bacterial M.HhaI enzyme has been considered as a stepwise nucleophilic addition of Cys-81-S- to cytosine C6 followed by C5 nucleophilic replacement of the methyl of S-adenosyl-L-methionine to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine.', 'Hydroxide at 10(-7) mole fraction (pH = 7) is shown to be sufficient for the required catalysis.', 'On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase.', 'We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis.', 'R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a.', 'While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.', 'These two methyltransferases recognize CpG and GCGC sequences, respectively, and transfer a methyl group to the C5 atom of cytosine (C).', 'Most prokaryotic (cytosine-5)-DNA methyltransferases increase the frequency of deamination at the cytosine targeted for methylation in vitro in the absence of the cofactor S-adenosylmethionine (AdoMet) or the reaction product S-adenosylhomocysteine (AdoHcy).', 'The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet.', 'These methylation patterns are established and maintained by DNA cytosine-5 methyltransferase (MTase), a approximately 1500 amino acid enzyme containing a regulatory N-terminal domain and a catalytic C-terminal domain.', 'These results lend strong support to a catalytic mechanism in which an enzyme sulfhydryl group undergoes Michael addition to the C5-C6 double bond, thus activating position C-5 of the substrate DNA cytosine residue for electrophilic attack by the methyl donor SAM.', "2'-Deoxyoligonucleotides with 5-fluorocytosine residues incorporated at specific positions of the nucleotide sequence are tools of great potential in the study of the catalytic mechanism by which DNA cytosine methyltransferases methylate the 5-position of DNA cytosine residues in specific sequence contexts.", 'The enzyme has Mr 130 000, and introduces methyl groups from S-adenosylmethionine into the 5 position of cytosines in DNA.']
['The catalytic mechanism of the DNA (Cytosine-5-)-methyltransferase involves nucleophilic attack of the C6 of the substrate cytosine by the single conserved cysteine of the enzyme, followed by C5 nucleophilic replacement of the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. It has been also demonstrated that Phe and Glu, which are found in the catalytic motifs I and II of the enzyme are important for AdoMet binding and catalysis.']
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