Structure, function and mechanism of exocyclic DNA methyltransferases

KINETIC STUDIES OF EXOCYCLIC DNA MTases

AdoMet-dependent DNA MTases are Bi Bi catalysts, and several kinetic mechanisms can be envisioned for an overall catalytic reaction (Figure 4). The substrate binding may be random (Figure 4A) or ordered (Figures 4B and 4C), and the release of products could be in an ordered or random manner as well. A Ping Pong mechanism or double-displacement mechanism is also possible in which one of the products is formed and released from the enzyme before the binding of other substrate. In this mechanism, the enzyme would be methylated in a first DNA-independent reaction and the methyl group would subsequently be transferred to the DNA in an AdoMet-independent reaction. The sequence of two methyl-transfer events would require stereochemical retentions of configuration at the methyl group. Studies carried out for the steric course of the methyl-transfer reaction using C⁵-cytosine MTase M.HhaI and an N⁶-adenine MTase M.EcoRI, showed that the methyl-transfer reaction proceeded with an inversion (S_N2 mechanism) of configuration [22]. Therefore these results argue for the absence of a Ping Pong mechanism, but suggest that a sequential mechanism is possible in which a ternary complex of the enzyme is formed. A limited number of DNA MTases have been characterized in terms of kinetic mechanisms. All C⁵-cytosine MTases studied so far follow an ordered Bi Bi mechanism with DNA binding first, followed by AdoMet binding (Figure 4C) [5]. The kinetic mechanisms for M.BamHI [23,24], a N⁴-cytosine MTase, and M.EcoP15I [25], M.EcoRI [26], M.RsrI [27], M.EcaI [28], CcrM [29], M.TaqI [30], M.EcoRV [31], T4 Dam [32], EcoDam [33,34] and M.KpnI [35], all N⁶-adenine MTases, have been elucidated. In general, N⁶-adenine MTases exhibit different kinetic mechanisms. Among the exocyclic adenine DNA MTases, an ordered reaction mechanism has been shown for M.EcoRI [26], M.EcoRV [31], T4 Dam [32] and M.KpnI [35], with the enzymes binding to AdoMet first (Figure 4B), whereas, in the case of CcrM [29], an ordered mechanism was postulated with the enzyme binding to DNA first (Figure 4C). A random mechanism of substrate binding has been reported for the EcoP15I MTase [25], M.EcaI [28] and M.BamHI [23,24] (Figure 4A). It is interesting that these MTases, which contain structurally similar domains, have diverse kinetic mechanisms. Table 1 summarizes the kinetic parameters obtained for all the exocyclic amino MTases studied so far.

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Figure 4 Sequential kinetic mechanisms observed with AdoMet-dependent DNA MTases

(A) Random Bi Bi mechanism. (B) Ordered mechanism, AdoMet binding first. (C) Ordered mechanism, DNA binding first. meDNA, methylated DNA.

The discrimination between DNA sequences is the key feature of DNA recognition and has been extensively investigated with DNA MTases. The interaction of the enzyme with DNA is complicated by the requirements for a chemical transformation of the target sequence. This means that enzymes cannot achieve specificity simply by increasing their affinity for cognate DNA sequences, as very tight binding would compromise catalysis. In general, all DNA MTases, including amino MTases, display very slow turnover or rate of methylation (Table 1). The low turnover coupled with their generally strong binding to their target DNA sequence means that k_cat/K_m values are high and that the MTases display fair-to-high specificity for methylation of their target sequences. How do DNA MTases discriminate between DNA sequences with such incredible specificity? All DNA MTases studied to date bind their cognate recognition sequences more strongly than to non-specific DNA, and AdoMet as well as its analogues, AdoHcy (S-adenosyl-L-homocysteine) and sinefungin increase the affinity of DNA MTases for their substrates and improve selectivity [47,55–60]. Despite the enhanced effectiveness of MTases on long DNA substrates, most DNA-binding studies have been performed using short DNA duplexes. Binding affinities for duplexes containing the target site are typically in the range 10⁻⁸–10⁻⁶ M (Table 2). Most studies use naturally occurring highly polymeric DNA as the substrate to determine the kinetic parameters of the reaction. The analysis of such data is complicated by the fact that many MTases are assumed to use the facilitated diffusion of the bound enzyme in a one-dimensional search for its specific recognition site. However, the use of relatively short oligonucleotide duplexes containing the specific recognition site simplifies the experimental conditions and allows one to obtain more accurate data to calculate reaction parameters.

In order to determine the rate-limiting step of the reaction catalysed by DNA MTase, the rate of accumulation of methylated DNA product is estimated using steady-state reaction conditions. When the progress curve of product formation over time is extrapolated back to the y-axis (time zero), the intercept is not zero. The non-zero intercept is most simply taken to indicate that the steady-state rate of catalysis is limited overall by a step in the kinetic pathway after the methyl group transfer step. Such bursts have been observed for M.EcoRI [67], M.PvuII [68] and T4 Dam [43], indicating that the release of products is the rate-limiting step in this reaction. In contrast, the rate of methyl group transfer was the rate-limiting step in the M.TaqI methylation reaction [30]. Reactions under single-turnover conditions ([E]⋙[S]) are performed in order to determine the rate of methylation (k_methylation). M.EcoRI catalyses a very rapid methyl group transfer with the k_methylation being 300-fold higher than the reaction rate constant (k_cat) [67]. On the other hand, the k_cat and k_methylation values for M.BamHI are 0.053 s⁻¹ and 0.07–0.1 s⁻¹ respectively [24]. Pre-steady-state kinetic analysis revealed in the case of T4 Dam that the k_methylation for unmethylated and hemimethylated substrates was at least 20-fold greater than the overall k_cat, indicating that the release of products is the rate-limiting step in the reaction [24].

By using a combination of pre-steady-state kinetics and spectroscopic approaches, it has been possible to put forward a detailed reaction pathway for M.EcoRI. The enzyme binds DNA at a rate of 1.6×10⁷ M⁻¹·s⁻¹ and then undergoes a slow isomerization to a catalytically competent form (E-DNA^s′). Catalysis of the methyl group transfer occurs next at a rate of 200 s⁻¹ to produce an enzyme–product complex. Enzyme-methylated DNA exists in two forms: (i) a slow dissociation complex (E-DNA^M*) which later isomerizes into (ii) the fast dissociation complex (E-DNA^M**). The methylated target adenine (DNA^M* and DNA^M**) remains in an extrahelical position in both these complexes even after its release from the enzyme. Restacking of the target base takes place at different rates: 0.6 s⁻¹ and 50 s⁻¹ for the fast and slow dissociation complexes respectively. Therefore the release of the methylated DNA appears to be the rate-limiting step [69,70]. In general, for the DNA MTases studied, except in the case of T4 Dam, the release of the methylated product often limits catalysis.

T4 Dam is similar to bacterial Type II DNA MTases with respect to its kinetic parameters, except that it has lower K_m values for substrates and a higher catalytic constant. The relatively high specificity constant of T4 Dam (k_cat/K_m=2.4×10⁶ M⁻¹·s⁻¹) indicates that it is one of the most efficient enzymes among several MTases studied so far (Table 1). On comparing the presteady-state and single-turnover methylation kinetics of T4 Dam, it was observed that T4 Dam–AdoMet functions as a monomer under steady-state conditions, whereas under single-turnover conditions, a catalytically active complex containing two T4 Dam–AdoMet molecules was observed initially, and two methyl groups were transferred per duplex [71]. It was also observed that preformed T4 Dam–AdoMet complexes were more efficient than preformed T4 Dam–DNA complexes in the first round of catalysis by pre-steady-state kinetics [71]. Based on these results, an ordered Bi Bi mechanism was proposed for T4 Dam where AdoMet bound first followed by DNA. The proposed mechanism was supported further by initial velocity-dependence studies, product-inhibition and substrate-inhibition studies, where AdoMet binds before DNA and methylated DNA is released first followed by AdoHcy [32]. However, the stimulatory effect of high AdoMet concentrations on methylation rate of DNA indicated that the mechanism was more complex. This effect could be explained if T4 Dam were to possess two AdoMet-binding sites. The first is the catalytic site with a high affinity for AdoMet, and the second is an allosteric site with a low affinity for AdoMet. The presence of two AdoMet-binding sites has been suggested for EcoDam and M.PvuII [57,68]. Free T4 Dam randomly interacts with substrates DNA and AdoMet to form a ternary T4 Dam–AdoMet–DNA complex in which T4 Dam undergoes a conformational change to attain an active form to carry out methylation. After the methyl group transfer from AdoMet to DNA, methylated DNA dissociates rapidly (1.7 s⁻¹) from the complex. In contrast, dissociation of AdoHcy proceeds relatively slowly (0.018 s⁻¹), indicating that release of AdoHcy is a rate-limiting step. The release of AdoHcy from enzyme–AdoHcy complex can occur by two pathways. The first involves release of AdoHcy, while T4 Dam remains in the active conformation and specifically binds to the second AdoMet molecule. Then, the T4 Dam–AdoMet complex binds to DNA for the next round of catalysis. The other proposed mechanism for methylation is where the release of AdoHcy is co-ordinated with the binding of the second AdoMet in a concerted manner, while T4 Dam remains in the active form. The resulting T4 Dam–AdoMet complex then binds to DNA for the next round of methylation. Thus the role of AdoMet in the methylation reaction of T4 Dam goes beyond that of simply serving as a methyl donor. As a consequence of the AdoMet-induced conformational rearrangement and increased specificity, T4 Dam is capable of undergoing a rapid reorientation to the productive strand in an asymmetrically modified recognition site [71]. While similar results were obtained for the M.RsrI [27] and EcoDam [33], Reich and Mashhoon [67], measuring single turnovers in the reaction catalysed by M.EcoRI, showed that there was no specificity in binding orientation, i.e. the enzyme methylated only 50% of the substrate–hemimethylated duplexes.

Pre-incubation studies carried out by Urig et al. [33] demonstrated that the preformed enzyme–DNA complex was the preferred way of complex assembly than pre-formed enzyme–AdoMet complex, because the rate of methylation was 10-fold higher for the pre-formed enzyme–DNA complex. On the basis of this result only, it was concluded that EcoDam followed an ordered Bi Bi mechanism where DNA binds first, followed by AdoMet. However, Mashhoon et al. [34] have argued that these results provide little evidence for any order of substrate addition. They provide direct evidence for a competent enzyme–AdoMet complex by isotope partitioning experiments. In addition, using steady-state kinetics and product inhibition studies, Mashhoon et al. [34] have shown that EcoDam follows an ordered Bi Bi mechanism where AdoMet binds first followed by DNA.

Motifs

The primary sequences of the C⁵-MTases share a set of conserved motifs (I–X) in a constant linear order (Figures 1 and 2) [1,8–10]. Several of these motifs are responsible for three basic functions: (i) AdoMet binding, (ii) sequence-specific DNA binding, and (iii) catalysis of methyl group transfer. Before any of the DNA MTases were characterized structurally, two motifs were assigned functional roles. It was proposed that motif I (FXGXG) was part of the AdoMet-binding site. This was based on the presence of motif I in a wide variety of AdoMet-dependent enzymes, including all DNA, RNA and several protein MTases [1,8,88–90]. In the case of C⁵-cytosine MTases (Figure 2A), it was shown that motif IV which possesses an invariant Pro-Cys dipeptide was involved in methyl group transfer [10,91,92]. Mutagenesis of this dipeptide, leading to loss of function in M.EcoRII, M.HhaI and M.HaeIII, was one of the several criteria proposed for the importance of the Pro-Cys dipeptide sequence in motif IV [10,91,92]. The region between motif VIII and motif IX was found to be highly diverse and was termed as a variable region. This region was shown to be involved in target DNA recognition and was aptly termed as TRD [93,94]. Initially only motifs I and IV could be recognized in the primary sequences of N⁴-cytosine and N⁶-adenine MTases. On the basis of this finding, Malone et al. [7] performed a structure-guided sequence comparison of N⁶-adenine and N⁴-cytosine DNA MTases. This analysis revealed that the N⁴-cytosine, N⁶-adenine and C⁵-cytosine MTases were closely related to one another. Nine conserved motifs, which correspond to motifs I–VIII and X found previously in C⁵-cytosine MTases, were identified in exocyclic MTases. The amino MTases have been subdivided further into three groups, namely α, β and γ, which are characterized by distinct linear orders for the conserved motifs [7]:

group α: N-terminus–AdoMet-binding region–TRD–catalytic region–C-terminus;

group β: N-terminus–catalytic region–TRD–AdoMet-binding region–C-terminus;

group γ: N-terminus–AdoMet-binding region–catalytic region–TRD–C-terminus.

Group α contains N⁶-adenine MTases (Figure 2B), group β contains both N⁴-cytosine MTases and N⁶-adenine MTases (Figure 2C), and group γ consists of M.TaqI and other N⁶-adenine MTases (Figure 2D). This grouping does not really demarcate N⁴-cytosine from N⁶-adenine MTases. The amino MTases belonging to the group γ have a motif order very similar to C⁵-cytosine MTases, except that they differ in the position of motif X. A homologue of motif IX in C⁵-cytosine MTases could not be identified easily in amino MTases.

A brief description of some of the motifs present in amino MTases is given below. Motif I, which is conserved among all AdoMet-dependent enzymes, accommodates the methionine moiety of AdoMet. It is analogous to the so-called G-loop in NAD⁺-binding enzymes with the consensus sequence GXGXXG. In α or β amino MTases, the consensus sequence contains a strictly conserved phenylalanine residue, whereas, in the γ amino MTases, this amino acid is missing. The role of motif I in some N⁶-adenine MTases has been assessed by site-directed mutagenesis. It was shown in M.EcoKI, a member of the Type I R-M systems, that substitution of aspartic acid for the first glycine residue in motif I completely abolished AdoMet binding, but did not alter the DNA-binding properties of the enzyme [95]. Substitution of arginine or serine for the second glycine residue in motif I of M.EcoP15I abolished AdoMet binding, leading to the loss of enzyme activity [96]. When Phe³⁹ in motif I of M.EcoRV was replaced with alanine, the mutant enzyme did not bind AdoMet, although DNA binding was not affected [97]. Motifs II and III are less conserved in amino MTases as compared with C⁵-cytosine MTases and are also associated with AdoMet binding. It has been proposed that the conserved charged amino acid (aspartic acid/glutamic acid) present in motif II interacts with the ribose hydroxy groups of AdoMet, whereas aspartic acid of motif III hydrogen bonds to adenine N⁶ position. The bulky hydrophobic side chains (part of motif III) make the van der Waals contacts with AdoMet's adenine. The backbone amide groups of amino acids in motif III also make hydrogen-bond contacts with N¹ of the adenine moiety of AdoMet. Substitution of alanine for Asp⁵⁸ (motif II) in M.EcoRV resulted in a mutant variant of M.EcoRV which was defective in AdoMet binding [97].

Motif IV, also called the DPPY motif, based on early sequence comparisons is the most significant among the conserved motifs with the consensus sequence (S,N/D)PP(Y/W/F). Both α and β N⁶-adenine MTases, with few exceptions, contain the consensus motif DPPY (Figures 1 and 2). The γ subgroup of N⁶-adenine MTases (M.TaqI) contains a NPPY sequence, whereas N⁴-cytosine MTases contain a SPPY sequence. Several groups have performed mutational studies on amino acids in motif IV which in turn have revealed the importance of this motif in catalysis [96–101]. Motif IV accommodates target adenine after it has flipped out of the DNA helix. The N⁶ amino group forms hydrogen bonds to the side chain of serine or aspartic acid and one of the backbone oxygen atoms [21]. This would polarize N⁶ negatively and activate it for direct transfer of the methyl group from AdoMet. In a random mutagenesis/selection study, Friedrich et al. [101] identified Asp¹⁹³ (motif IV) in M.EcoRV as being a very important residue in catalysis, although the mutant D193G was able to bind DNA. Roth et al. [97] have constructed variants of M.EcoRV where aspartic acid of motif IV was replaced with either asparagine or alanine. Both of these variants were catalytically inactive, but were able to bind DNA and AdoMet with similar affinities as the wild-type M.EcoRV. Results with the D193N and D193A variants of M.EcoRV are in agreement with mutational data obtained with M.EcoKI, M.BcgI and EcoDam (DNA adenine MTase) [95,52,99]. In M.EcoKI, an NPPF to DPPF exchange resulted in a catalytically inactive enzyme that could still bind the AdoMet [95]. Variants of M.BcgI in which the NPPY asparagine residue was replaced by alanine, aspartic acid or glutamine were all catalytically inactive [52]. Motif IV has been proposed to be analogous to the catalytic PCQ motif of C⁵-cytosine MTases. From the three-dimensional structures, it has been shown that the conserved NPPY motif in M.TaqI, an N⁶-adenine MTase, overlaps with the PCQ motif of M.HhaI, a C⁵-cytosine MTase, almost exactly when the two structures are superimposed [4,102,103]. In addition to aspartic acid, tyrosine is the second conserved amino acid residue in motif IV that has been investigated in detail. Pues et al. [49] have shown in the case of M.TaqI that replacement of Tyr¹⁰⁸ with phenylalanine or tryptophan led to mutant MTases with almost identical or somewhat reduced enzymatic activities, compared with the wild-type M.TaqI. In contrast, the replacement of Tyr¹⁰⁸ with alanine or glycine resulted in mutant MTases with dramatically reduced enzymatic activities which clearly demonstrates the importance of an aromatic amino acid side chain in motif IV for enzymatic activity. Similar results have been obtained with M.EcoRV [97] and M.EcoKI [95]. However, in the case of M.EcoP15I and M.EcoP1I, DPPY to DPPW exchange inactivated the enzyme [96,100].

In group γ, motif V contains the consensus sequence (N/D)LYXXF(L/V/I). The phenylalanine residue in motif V interacts through its aromatic side chain with the adenine of AdoMet. Schluckebier et al. [102] have shown that Leu¹⁴² (part of motif V) in M.TaqI makes van der Waals contacts with the adenine of AdoMet. It was therefore suggested in the case of groups α and β that one of the conserved hydrophobic side chains in motif V may have a same spatial position as the leucine residue in group γ MTases. Amino acids in motif VI constitute the C-terminal residues which are part of target adenine-binding site, whereas the arginine residue in motif VII is located in the putative DNA-recognition cleft possibly functioning in DNA binding. Ser²²⁹ of M.EcoRV, which is highly conserved in amino MTases, is located in motif VI and corresponds to Glu¹¹⁹ in M.HhaI. This residue is involved in acid–base catalysis in M.HhaI. Val-Phe, the consensus sequence of motif VIII forms the lower part of the target adenine-binding site and borders the DNA-binding cleft. The phenylalanine residue in this motif is well conserved (sometimes as tyrosine or tryptophan) in all amino MTases. This aromatic residue seems to make favourable contacts with the target DNA adenine in the case of M.TaqI. The high degree of conservation and the positioning of the side chain in the centre of the active site suggest that this aromatic amino acid holds a key role in catalysis [17]. In M.EcoRV, it has been shown clearly that Tyr²⁵⁸ (motif VIII) provides an aromatic ring and/or hydroxy group to interact with the adenine base, since the Y258A variant is catalytically almost inactive, but binds to DNA and AdoMet, although with reduced affinity for the AdoMet [97]. In M.TaqI, when Phe¹⁹⁶ (motif VIII) was replaced with alanine, there was a significant decrease in the catalytic constant, whereas the F196W variant had almost wild-type catalytic activity, clearly indicating the importance of an aromatic amino acid in this position [49]. There is a marked difference in the position of motif X in the primary amino acid sequence of amino MTases and C⁵-cytosine MTases. This motif is expected to form a helix next to the β-strand formed by motif I. Cheng [4] suggested that motif X, along with motif I and motif IV, provides the binding pocket for AdoMet's methionine moiety.

Structures

Although several DNA MTases have been sequenced and purified, only a handful of these MTases have been crystallized and their three-dimensional structures determined. M.HhaI [76], M.HaeIII [104] and human DNMT2 [105], belonging to the C⁵-cytosine MTases, are the only three DNA MTases whose three-dimensional structures were solved in the presence of DNA. The three dimensional structures of six N⁶-adenine MTases are known: M.TaqI [19,20], M.DpnM [106], M.RsrI [107,108], M.MboIIA [109], T4 Dam [110–112] and EcoDam [85].

In general, MTases are bilobal structures folded into two domains: a larger catalytic domain with both the active site for methyl transfer and the AdoMet-binding site and a smaller target (DNA)-recognition domain that possesses loops implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base. The DNA is bound in the cleft between the two domains flanked by several loops. The importance of some amino acid residues located in these loops has been confirmed by several site-directed mutagenesis studies.

The large domain of all DNA MTases share a common structural core which consists of a six-stranded parallel β-sheet with a seventh strand inserted in an antiparallel fashion between the fifth and sixth strands (Figure 5). Within the large domain, one can clearly see two subdomains. One of the subdomains creates the AdoMet-binding site and the other subdomain is the binding site for the extrahelical target base. The large subdomain contains the nine characteristic amino acid motifs. The small domains of different DNA MTases are not similar in amino acid sequence, size or structure, and therefore cannot be superimposed.

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Figure 5 Structures of exocyclic DNA MTases

(A) Three-dimensional structure of M.TaqI with DNA (PDB code 1G38). The DNA backbone in the structure is shown as arrows and base pairs are shown as blue rings. The nucleotide shown in blue and present within the protein back bone is the flipped target adenine. Tyr¹⁰⁸ and Phe¹⁹⁶ shown in yellow are present in the vicinity of flipped target adenine. (B) The crystal structure of two molecules of T4 Dam bound to a palindromic 12 bp DNA duplex in presence of AdoHcy (PDB code 1Q0T). The DNA backbone in the structure is shown as tubes, base pairs are shown as sticks and AdoHcy molecules are shown as space-filled models. (C) The crystal structure of DpnM with AdoMet (PDB code 2DPM). The AdoMet molecule in the structure is shown as a ball and stick model. (D) Three-dimensional structure of M.PvuII in presence of AdoHcy (PDB code 1B00). The AdoHcy molecule in the structure is shown as a ball and stick representation. (E) The crystal structure of M.RsrI in presence of AdoMet (PDB code 1NW5). The AdoMet molecule in the structure is shown as a ball and stick model. The two amino acids (Leu⁷² and Asp¹⁷³) are represented as space-fill models. (F) The crystal structure of MboIIA in the presence of AdoMet (PDB code 1G60). The AdoMet molecule in the structure shown in space-fill representation.

All of these enzymes share a remarkably similar catalytic domain structure, resembling an α/β Rossman fold with conserved binding patterns for the cofactor AdoMet and modified base corresponding mainly to conserved motifs I and IV. In all cases, the substrate to be methylated is bound or expected to bind in a pocket adjacent to the AdoMet-binding site, which is formed by different amino acids in different MTases. Beyond the shared catalytic MTase fold, the active sites of M.HhaI, M.TaqI, M.PvuII and M.RsrI are superimposable. The proposed catalytic function of motif IV in the amino MTases is to activate the exocyclic amine nucleophile by donating the hydrogen bonds from side chain (serine or aspartic acid) and backbone oxygen atoms [21]. The M.TaqI–DNA co-crystal structure confirms this proposal. The invariant DPPY of T4 Dam is superimposable on to the corresponding motif in the ternary complex of M.TaqI [112], as well as the binary complex of M.DpnII [106] and M.RsrI [107,108] in the absence of DNA, suggesting that the conformation of DPPY is quite stable and highly conserved.

Structural studies have provided evidence that the aspartic acid/aspargine/serine residue of motif IV [(D/N/S)PP(Y/F)] contacts the exocyclic amino group of the flipped base and activates it for methyl group transfer and the aromatic amino acid residue stacks to the flipped target base and stabilizes the flipped conformation. The structures of DNA MTases suggest that the flipped state is locked by structural rearrangements of the enzyme and DNA, which prevent back rotation of the flipped base.

The AdoMet-binding site is significantly conserved in all DNA MTases. In general, it is created by residues from the motifs I–III and X, which form conserved contacts to almost every hydrogen-bond donor and acceptor of AdoMet and, in addition, several hydrophobic interactions to the cofactor. The roles of many of these residues have been confirmed by mutagenesis studies. A V-shaped cleft in the molecule accommodates AdoMet in an extended conformation. In one arm of the V-shaped cleft, adenosine is located, while a sulfonium group (S^δ+) occupies the tip of the cleft. The other arm harbours the methionine moiety. The positively charged amino acid residues in the loop region (connecting N- and C-terminal domains) are in position to interact with the phosphate groups of the DNA backbone. Interestingly, more than one AdoMet-binding site has been observed in M.PvuII [68], EcoDam [56,57] and T4 Dam [112,113]. The significance of the second AdoMet molecule is not clear. It has been suggested that the second AdoMet molecule (i) increases the occupancy of catalytically relevant AdoMet-binding site, and (ii) confers additional sequence specificity. Also, if the binding pocket for the methylatable base were occupied by the second AdoMet, base flipping would presumably require displacement of AdoMet and this could reduce the effective affinity for non-specific sites [68,83].

The structure of M.TaqI in complex with AdoMet, AdoHcy and with the competitive inhibitor sinefungin was determined at a resolution of 2.4 Å (1 Å=0.1 nm) [19,20,66]. M.TaqI binds to adenosine moieties of AdoMet, AdoHcy and sinefungin in a similar manner, but the binding is different for their amino acid moieties [66]. The three-dimensional structure of M.TaqI with a specific 10 bp hemimethylated DNA and a non-reactive analogue 5′-[2(amino)ethylthio]-5′-deoxyadenosine showed that the target adenine present in the recognition sequence is indeed rotated out of the double helix to transfer the methyl group (Figure 5A) [20]. In the active site, two aromatic amino acid residues, Tyr¹⁰⁸ (motif IV) and Phe¹⁹⁶ (motif XIII) bind to the flipped target base, and the torsion of the methionine moiety of AdoMet by M.TaqI brings the methyl group within the reach of the flipped target base to transfer the methyl group. Both Tyr¹⁰⁸ and Phe¹⁹⁶ probably hold an important role in transfer of the methyl group by decreasing the energy of the positively charged transition state via cation–π interaction [17].

The ternary complex of T4 Dam with AdoHcy and 12 bp duplex revealed a non-specific loose DNA-binding mode with two Dam monomers bound to one duplex (Figure 5B). One T4 Dam binds to a single DNA duplex spanning 7 bp, whereas the other T4 Dam binds in the joint of two DNA molecules spanning 6 bp. There are no significant conformational changes in T4 Dam after non-specific binding in the ternary complex. However, the orientations of the two molecules of T4 Dam relative to the DNA helical axis are different. The first T4 Dam molecule is rotated by ∼20 Å relative to the second T4 Dam molecule, resulting in a larger cavity for first one compared with the second T4 Dam [107–109]. Three structures for T4 Dam in ternary complexes with partially and fully specific DNA and a methyl donor analogue have been described. These structures illustrate the transition in enzyme–DNA interactions from non-specific to specific interactions, suggesting that there is a temporal order for formation of specific contacts. It is clear from such structural studies that T4 Dam moves along DNA and rotates up and down as a rigid body relative to the DNA. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.

The ternary structure of EcoDam in complex with cognate DNA is similar in many ways to the T4 Dam complex with cognate DNA. While the first base pair is contacted by Lys⁹ in EcoDam, Arg¹³⁰ interacts with first guanine in T4 Dam. Interestingly, the flipped target adenine binds to the surface of EcoDam in the absence of AdoMet (a possible intermediate in the base flipping pathway) and the orphaned thymine displaying structural flexibility [85].

Among the exocyclic amino MTases, M.PvuII [21] is the only N⁴-cytosine MTase whose three-dimensional structure is known (Figure 5D). Interestingly, in the structure of the M.PvuII–AdoMet complex, an extra electron density was found near the first AdoMet molecule. This density fitted for an adenosine moiety of AdoMet with the methionine moiety extending into the solvent.

Thomas et al. [108] determined the three-dimensional structures of M.RsrI in the presence of AdoMet, AdoHcy and sinefungin (Figure 5E). Two distinct binding conformations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group towards the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. The two different modes of ligand binding by M.RsrI may be due to the differences in the positive charges on the ligands. The sulfur of AdoMet and the ϵ-amino group of sinefungin carry a formal positive charge, whereas AdoHcy is uncharged at the analogous position.

Szegedi and Gumport [54] observed that two mutants of M.RsrI (L72P and D173A) had altered catalytic and DNA-binding properties. The M.RsrI structure does provide an explanation for the functional defects of these mutants [107]. Leu⁷² is located in 3₁₀-helix D1, which is C-terminal to the catalytic motif, DPPY. Mutation of this residue to proline destabilizes helix formation of D1 or alters the flexibility of β-hairpin loop. This in turn results in the disruption of the normal conformation of the DPPY motif. The crystal structure of L72P was solved by Thomas et al. [108] and it showed that mutant M.RsrI did not co-crystallize with the cofactor. Substitution of Leu⁷² by proline caused a kink in the loop resulting in a compression of the loop on one side and the end of an α-helix shifted on the other side.