Structure, function and mechanism of exocyclic DNA methyltransferases

DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an 'AdoMet-dependent MTase fold'. DNA methylation has been reported to be essential for bacterial virulence, and it has been suggested that DNA adenine MTases (Dams) could be potential targets for both vaccines and antimicrobials. Drugs that block Dam could slow down bacterial growth and therefore drug-design initiatives could result in a whole new generation of antibiotics. The transfer of larger chemical entities in a MTase-catalysed reaction has been reported and this represents an interesting challenge for bio-organic chemists. In general, amino MTases could therefore be used as delivery systems for fluorescent or other reporter groups on to DNA. This is one of the potential applications of DNA MTases towards developing non-radioactive DNA probes and these could have interesting applications in molecular biology. Being nucleotide-sequence-specific, DNA MTases provide excellent model systems for studies on protein-DNA interactions. The focus of this review is on the chemistry, enzymology and structural aspects of exocyclic amino MTases.     

Synthesis of S-adenosyl-L-methionine analogs and their use for sequence-specific transalkylation of DNA by methyltransferases

Here we describe a one-step synthetic procedure for the preparation of S-adenosyl-L-methionine (AdoMet) analogs with extended carbon chains replacing the methyl group. These AdoMet analogs function as efficient cofactors for DNA methyltransferases (MTases), and we provide a protocol for sequence-specific transfer of extended side chains from these AdoMet analogs to DNA by DNA MTases. Direct chemoselective allylation or propargylation of S-adenosyl-L-homocysteine (AdoHcy) at sulfur is achieved under the acidic conditions needed to protect other nucleophilic positions in AdoHcy. The unsaturated bonds in beta position to the sulfonium center of the resulting AdoMet analogs are designed to stabilize the transition state formed upon DNA MTase-catalyzed nucleophilic attack at the carbon next to the sulfonium center and lead to efficient transfer of the extended side chains to DNA. Using these protocols, sequence-specific functionalized DNA can be obtained within one to two weeks.     

Is the HemK family of putative S-adenosylmethionine-dependent methyltransferases a "missing" zeta subfamily of adenine methyltransferases? A hypothesis

Previous comparative studies revealed close similarity among various groups of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases), indicating their common evolutionary origin. We present evidence for a remarkable similarity between the sequence and predicted structure of HemK (a widespread family of putative proteins encoded in genomes from bacteria to humans) and the catalytic domain of the gamma-subfamily of adenine-specific DNA MTases (N6mA MTases). We predict the structure and function of the putative catalytic domain of HemK proteins and speculate that the target-recognizing function may be conferred by the N-terminal variable region.     

Mechanisms for auto-inhibition and forced product release in glycine N-methyltransferase: crystal structures of wild-type, mutant R175K and S-adenosylhomocysteine-bound R175K enzymes

Glycine N-methyltransferase (S-adenosyl-l-methionine: glycine methyltransferase, EC 2.1.1.20; GNMT) catalyzes the AdoMet-dependent methylation of glycine to form sarcosine (N-methylglycine). Unlike most methyltransferases, GNMT is a tetrameric protein showing a positive cooperativity in AdoMet binding and weak inhibition by S-adenosylhomocysteine (AdoHcy). The first crystal structure of GNMT complexed with AdoMet showed a unique "closed" molecular basket structure, in which the N-terminal section penetrates and corks the entrance of the adjacent subunit. Thus, the apparent entrance or exit of the active site is not recognizable in the subunit structure, suggesting that the enzyme must possess a second, enzymatically active, "open" structural conformation. A new crystalline form of the R175K enzyme has been grown in the presence of an excess of AdoHcy, and its crystal structure has been determined at 3.0 A resolution. In this structure, the N-terminal domain (40 amino acid residues) of each subunit has moved out of the active site of the adjacent subunit, and the entrances of the active sites are now opened widely. An AdoHcy molecule has entered the site occupied in the "closed" structure by Glu15 and Gly16 of the N-terminal domain of the adjacent subunit. An AdoHcy binds to the consensus AdoMet binding site observed in the other methyltransferase. This AdoHcy binding site supports the glycine binding site (Arg175) deduced from a chemical modification study and site-directed mutagenesis (R175K). The crystal structures of WT and R175K enzymes were also determined at 2.5 A resolution. These enzyme structures have a closed molecular basket structure and are isomorphous to the previously determined AdoMet-GNMT structure. By comparing the open structure to the closed structure, mechanisms for auto-inhibition and for the forced release of the product AdoHcy have been revealed in the GNMT structure. The N-terminal section of the adjacent subunit occupies the AdoMet binding site and thus inhibits the methyltransfer reaction, whereas the same N-terminal section forces the departure of the potentially potent inhibitor AdoHcy from the active site and thus facilitates the methyltransfer reaction. Consequently GNMT is less active at a low level of AdoMet concentration, and is only weakly inhibited by AdoHcy. These properties of GNMT are particularly suited for regulation of the cellular AdoMet/AdoHcy ratio.     

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.     

The DNA and S-adenosylmethionine-binding regions of EcoDam and related methyltransferases

Previous comparison of the amino acid sequences of the GATC-methylating Escherichia coli Dam methyltransferase (MTase) with those of other adenine MTases (M.EcoRV, M.DpnII and T4Dam) localized four conserved regions. Regions III and IV have similarities with many other MTases. The sequence DPPY (or NPPY) is always present in region IV. It was suggested to be the AdoMet binding site. Publication of the nucleotide and amino acid sequences of M.CviBIII, M.DpnA and MutH give further credence to this assignment: M.DpnA, which also methylates GATC, has strong similarities with regions III and IV; M.CviBIII, a cytosine methylase, has a characteristic NPPY sequence in region IV, and only limited resemblance in region III; MutH, the GATC-specific endonuclease in DNA mismatch repair, has significant similarities uniquely in region III. The presently available evidence suggests that region III is the GAT(C) binding site and region IV is the AdoMet binding site. This hypothesis is strengthened by recent genetic findings.     

Sequence analysis and structure prediction of aminoglycoside-resistance 16S rRNA:m7G methyltransferases

Methylation of G1405 within bacterial 16S ribosomal RNA results in high-level resistance to specific combinations of aminoglycoside antibiotics. Only a few closely related methyltransferases (MTases), which carry out the respective modification (here dubbed "Agr", for aminoglycoside resistance), are known. It is not clear, whether they are related to "typical" S-adenosylmethionine (AdoMet)-dependent MTases or not. Demydchuk et al., 1998 proposed that the cofactor-binding region is localized at the C-terminus of Agr MTases, which implies an interesting case of sequence permutation. Since the Agr MTases lack significant sequence similarity to other proteins, we tested that hypothesis using more sensitive sequence/structure threading approach. Structure prediction confirmed the presence of a putative AdoMet-binding site in these proteins, albeit at a distinct location, resembling that of "typical", non-permuted MTases. Additionally, a small alpha-helical domain dissimilar to other proteins in the database was identified in the N-terminal region of Agr MTases. Comparison of a three-dimensional model of the Agr family member with a recently solved structure of reovirus mRNA capping MTase suggests that the mechanism of guanine-N7 methylation in rRNA and mRNA may be different.     

Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

KpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5'-GGTACC-3' and transfers the methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation and in vitro restriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.     

DNA methyltransferases: mechanistic models derived from kinetic analysis

The sequence-specific transfer of methyl groups from donor S-adenosyl-L-methionine (AdoMet) to certain positions of DNA-adenine or -cytosine residues by DNA methyltransferases (MTases) is a major form of epigenetic modification. It is virtually ubiquitous, except for some notable exceptions. Site-specific methylation can be regarded as a means to increase DNA information capacity and is involved in a large spectrum of biological processes. The importance of these functions necessitates a deeper understanding of the enzymatic mechanism(s) of DNA methylation. DNA MTases fall into one of two general classes; viz. amino-MTases and [C5-cytosine]-MTases. Amino-MTases, common in prokaryotes and lower eukaryotes, catalyze methylation of the exocyclic amino group of adenine ([N6-adenine]-MTase) or cytosine ([N4-cytosine]-MTase). In contrast, [C5-cytosine]-MTases methylate the cyclic carbon-5 atom of cytosine. Characteristics of DNA MTases are highly variable, differing in their affinity to their substrates or reaction products, their kinetic parameters, or other characteristics (order of substrate binding, rate limiting step in the overall reaction). It is not possible to present a unifying account of the published kinetic analyses of DNA methylation because different authors have used different substrate DNAs and/or reaction conditions. Nevertheless, it would be useful to describe those kinetic data and the mechanistic models that have been derived from them. Thus, this review considers in turn studies carried out with the most consistently and extensively investigated [N6-adenine]-, [N4-cytosine]- and [C5-cytosine]-DNA MTases.     

Dimethyl sulfoxide stimulates the catalytic activity of de novo DNA methyltransferase 3a (Dnmt3a) in vitro

Mammalian DNA methyltransferase Dnmt3a is required for de novo methylation of CpG dinucleotides in genomic DNA. While DNA methyltransferase inhibitors have been extensively utilized both in vitro and in vivo, no stimulator of catalytic activity has been identified thus far. Here we show that the methyltransfer activity of Dnmt3a is stimulated by the addition of dimethyl sulfoxide (DMSO) to the reaction solution in vitro. Enzymatic analysis of initial reaction velocity suggests that the DMSO stimulation effect depends on the interaction between DMSO and the reaction substrates (DNA and AdoMet), but not the enzyme itself.     

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX,the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-l-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K_d = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.     

Characterization of the cofactor-binding site in the SPOUT-fold methyltransferases by computational docking of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine to three crystal structures

Background  

There are several evolutionarily unrelated and structurally dissimilar superfamilies of S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases). A new superfamily (SPOUT) has been recently characterized on a sequence level and three structures of its members (1gz0, 1ipa, and 1k3r) have been solved. However, none of these structures include the cofactor or the substrate. Due to the strong evolutionary divergence and the paucity of experimental information, no confident predictions of protein-ligand and protein-substrate interactions could be made, which hampered the study of sequence-structure-function relationships in the SPOUT superfamily.     

Crystal structure of MboIIA methyltransferase

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.     

AdoMet-dependent methylation, DNA methyltransferases and base flipping

Twenty AdoMet-dependent methyltransferases (MTases) have been characterized structurally by X-ray crystallography and NMR. These include seven DNA MTases, five RNA MTases, four protein MTases and four small molecule MTases acting on the carbon, oxygen or nitrogen atoms of their substrates. The MTases share a common core structure of a mixed seven-stranded beta-sheet (6 downward arrow 7 upward arrow 5 downward arrow 4 downward arrow 1 downward arrow 2 downward arrow 3 downward arrow) referred to as an 'AdoMet-dependent MTase fold', with the exception of a protein arginine MTase which contains a compact consensus fold lacking the antiparallel hairpin strands (6 downward arrow 7 upward arrow). The consensus fold is useful to identify hypothetical MTases during structural proteomics efforts on unannotated proteins. The same core structure works for very different classes of MTase including those that act on substrates differing in size from small molecules (catechol or glycine) to macromolecules (DNA, RNA and protein). DNA MTases use a 'base flipping' mechanism to deliver a specific base within a DNA molecule into a typically concave catalytic pocket. Base flipping involves rotation of backbone bonds in double-stranded DNA to expose an out-of-stack nucleotide, which can then be a substrate for an enzyme-catalyzed chemical reaction. The phenomenon is fully established for DNA MTases and for DNA base excision repair enzymes, and is likely to prove general for enzymes that require access to unpaired, mismatched or damaged nucleotides within base-paired regions in DNA and RNA. Several newly discovered MTase families in eukaryotes (DNA 5mC MTases and protein arginine and lysine MTases) offer new challenges in the MTase field.     

Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome

Mammalian DNA methyltransferases methylate cytosine residues within CG dinucleotides. By statistical analysis of published data of the Human Epigenome Project we have determined flanking sequences of up to +/-four base-pairs surrounding the central CG site that are characteristic of high (5'-CTTGCGCAAG-3') and low (5'-TGTTCGGTGG-3') levels of methylation in human genomic DNA. We have investigated the influence of flanking sequence on the catalytic activity of the Dnmt3a and Dnmt3b de novo DNA methyltransferases using a set of synthetic oligonucleotide substrates that covers all possible +/-1 flanks in quantitative terms. Methylation kinetics experiments revealed a >13-fold difference between the preferred (RCGY) and disfavored +/-1 flanking base-pairs (YCGR). In addition, AT-rich flanks are preferred over GC-rich ones. These experimental preferences coincide with the genomic methylation patterns. Therefore, we have expanded our experimental analysis and found a >500-fold difference in the methylation rates of the consensus sequences for high and low levels of methylation in the genome. This result demonstrates a very pronounced flanking sequence preference of Dnmt3a and Dnmt3b. It suggests that the methylation pattern of human DNA is due, in part, to the flanking sequence preferences of the de novo DNA MTases and that flanking sequence preferences could be involved in the origin of CG islands. Furthermore, similar flanking sequence preferences have been found for the stimulation of the immune system by unmethylated CGs, suggesting a co-evolution of DNA MTases and the immune system.     

DNA methyltransferases of the cyanobacterium Anabaena PCC 7120

From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium ANABAENA PCC 7120 has been deduced. ANABAENA has nine DNA MTases. Four are associated with Type II restriction enzymes (AVAI, AVAII, AVAIII and the newly recognized inactive AVAIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AVAVI, DmtB/M.AVAVII, DmtC/M. AVAVIII and DmtD/M.AVAIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AVAV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.     

DNA (cytosine-N4-)- and -(adenine-N6-)-methyltransferases have different kinetic mechanisms but the same reaction route. A comparison of M.BamHI and T4 Dam

We studied the kinetics of methyl group transfer by the BamHI DNA-(cytosine-N(4)-)-methyltransferase (MTase) from Bacillus amyloliquefaciens to a 20-mer oligodeoxynucleotide duplex containing the palindromic recognition site GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the K(m) for either DNA (100-fold higher) or S-adenosyl-l-methionine (AdoMet) (20-fold higher); this indicates that dead-end complexes did not form in the course of the methylation reaction. The DNA methylation rate was analyzed as a function of both substrate and product concentrations. It was found to exhibit product inhibition patterns consistent with a steady state random bi-bi mechanism in which the dominant order of substrate binding and product release (methylated DNA, DNA(Me), and S-adenosyl-l-homocysteine, AdoHcy) was Ado-Met DNA DNA(Me) AdoHcy. The M.BamHI kinetic scheme was compared with that for the T4 Dam (adenine-N(6)-)-MTase. The two differed with respect to an effector action of substrates and in the rate-limiting step of the reaction (product inhibition patterns are the same for the both MTases). From this we conclude that the common chemical step in the methylation reaction, methyl transfer from AdoMet to a free exocyclic amino group, is not sufficient to dictate a common kinetic scheme even though both MTases follow the same reaction route.     

Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases

Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.     

Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase

DNA methylation plays important roles via regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigm bacterial methyltransferase (MTase) HhaI (M.HhaI) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) onto the target cytosine in DNA, yielding 5-methylcytosine and S-adenosyl-L-homocysteine (AdoHcy). The turnover rate (k cat) of M.HhaI, and the other two cytosine-5 MTases examined, is limited by a step subsequent to methyl transfer; however, no such step has so far been identified. To elucidate the role of cofactor interactions during catalysis, eight mutants of Trp41, which is located in the cofactor binding pocket, were constructed and characterized. The mutants show full proficiency in DNA binding and base-flipping, and little variation is observed in the apparent methyl transfer rate k chem as determined by rapid-quench experiments using immobilized fluorescent-labeled DNA. However, the Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher K(AdoMet)D and K(AdoMet)M) leading to a faster turnover of the enzyme (10-fold higher k cat). Our analysis indicates that the rate-limiting breakdown of a long-lived ternary product complex is initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme.