J. Mol. Biol. (1995) 253, 618–632

Structure-guided Analysis Reveals Nine SequenceMotifs Conserved among DNA Amino-methyl-transferases, and Suggests a Catalytic Mechanismfor these Enzymes

Thomas Malone 1, Robert M. Blumenthal 2* and Xiaodong Cheng 1*

Previous X-ray crystallographic studies have revealed that the catalytic1W. M. Keck StructuralBiology Laboratory, Cold domain of a DNA methyltransferase (Mtase) generating C5-methylcytosine

bears a striking structural similarity to that of a Mtase generatingSpring Harbor LaboratoryCold Spring Harbor, NY N6-methyladenine. Guided by this common structure, we performed a

multiple sequence alignment of 42 amino-Mtases (N6-adenine and11724, USAN4-cytosine). This comparison revealed nine conserved motifs, correspond-2Department of Microbiology ing to the motifs I to VIII and X previously defined in C5-cytosine Mtases.

Medical College of Ohio The amino and C5-cytosine Mtases thus appear to be more closely relatedToledo, OH 43699-0008 than has been appreciated. The amino Mtases could be divided into threeUSA groups, based on the sequential order of motifs, and this variation in order

may explain why only two motifs were previously recognized in the aminoMtases. The Mtases grouped in this way show several other group-specificproperties, including differences in amino acid sequence, molecular massand DNA sequence specificity. Surprisingly, the N4-cytosine andN6-adenine Mtases do not form separate groups. These results haveimplications for the catalytic mechanisms, evolution and diversification ofthis family of enzymes. Furthermore, a comparative analysis of theS-adenosyl-L-methionine and adenine/cytosine binding pockets suggeststhat, structurally and functionally, they are remarkably similar to oneanother.

7 1995 Academic Press Limited

Keywords: amino acid sequence motif; catalytic mechanism; DNA*Corresponding authors methyltransferase; S-adenosyl-L-methionine; structural similarity

Introduction

DNA Mtases transfer methyl groups fromS-adenosyl-L-methionine (AdoMet) to specific pos-itions on bases in double-stranded DNA. The DNAMtases fall into two major classes, defined by theposition methylated. The members of one classmethylate a pyrimidine ring carbon yieldingC5-methylcytosine (5mC; e.g. HhaI Mtase, M.HhaI).Members of the second class methylate exocyclicamino nitrogens, forming either N6-methyladenine(N6mA; e.g. M.TaqI) or N4-methylcytosine (N4mC;

e.g. M.PvuII). Mtases of the two classes wereexpected to be substantially different from oneanother, based on the fact that their targets ofmethyl transfer are very different. This substratedifference can be illustrated by the respectives-charge densities for the methyl-replaceable hydro-gen atoms, which are +0.22e on the exocyclic aminogroups of both adenine and cytosine, and −0.03e onthe 5-carbon of cytosine (Renugopalakrishnan et al.,1971). Do Mtases from the two classes, in fact, differsubstantially from one another?

Analysis of gene sequences has suggested thatthe two Mtase classes are quite different. Allbacterial 5mC Mtases, and a Chlorella virus 5mCMtase, contain a set of ten conserved blocks ofamino acid residues (I through X: Posfai et al., 1989;Cheng et al., 1993a; Kumar et al., 1994; Lauster et al.,1989; Som et al., 1987). These conserved motifshave the same linear order, which simplifies theiridentification in primary sequences. These ten con-

Abbreviations used: AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine;Mtase, methyltransferase; 5mC, C5-methylcytosine;N4mC, N4-methylcytosine; N6mA, N6-methyladenine;amino Mtase; Mtase generating N4mC or N6mA;COMtase, catechol O-methyltransferase; CM, conservedmotif; vdw, van der Waals.

0022–2836/95/440618–15 $12.00/0 7 1995 Academic Press Limited

DNA Amino-methyltransferases 619

served motifs are even present in the carboxy-terminal 0500 amino acid residues of the mouse,human, and Arabidopsis CpG 5mC Mtases (Bestoret al., 1988; Scheidt et al., 1991; Finnegan & Dennis,1993; Guenthner et al., 1992). In contrast, linearalignment of the amino acid sequences of the aminoMtases has not revealed such conservation (seebelow).

Before any DNA Mtases had been characterizedstructurally, two motifs of 5mC Mtases wereassigned functional roles. Motif I (the core of whichis almost always a Gly-rich sequence, such asAla19-Gly-Leu-Gly-Gly in M.HhaI) was presumedto be part of the AdoMet binding site. Thisassignment was based on the presence of thisGly-rich sequence in a wide variety of AdoMet-de-pendent Mtases in addition to the 5mC Mtases,including N6mA and N4mC DNA Mtases, andRNA, protein, and small molecule Mtases (Kli-masauskas et al., 1989; Ingrosso et al., 1989; Smithet al., 1990; Wilson & Murray, 1991; Kagan & Clarke,1994). The other motif to which a role could beassigned was motif IV, which contains an invariantdipeptide (Pro-Cys). The Cys in this motif is theactive site nucleophile, and forms a transientcovalent bond to the 6-carbon of the methylatablecytosine (Santi et al., 1983, 1984; Wu & Santi, 1987;Chen et al., 1991; Friedman & Ansari, 1992; Smithet al., 1992; Wyszynski et al., 1992; Hanck et al., 1993;Mi & Roberts, 1993; Chen et al., 1993). Most aminoMtases lack a Pro-Cys dipeptide.

Structural analysis, however, has found strikingsimilarity between DNA Mtases of the two classes.Information on the structures of DNA Mtases firstcame from studies of M.HhaI and M.TaqI, which,like most Mtases from type II restriction-modifi-cation systems, are active as monomeric enzymes.These studies have provided insights into Mtasedomain organization and its relationship to theconserved sequence motifs (Cheng et al., 1993a;Labahn et al., 1994). The structure of an M.HhaI–DNA complex provided further insight into thefunctions of several conserved amino acids impli-

cated in DNA sequence specificity, catalysis andAdoMet binding (Cheng et al., 1993b; Klimasauskaset al., 1994). M.HhaI is folded into two broaddomains: a catalytic domain that contains the activesite and AdoMet-binding regions, and a DNA-rec-ognition region. The structure of M.TaqI complexedwith AdoMet is also bilobal (Labahn et al., 1994).This bilobal structure may be a general property ofDNA Mtases, as it has also been seen followinglimited proteolysis of M.EcoRI (Reich et al., 1991)and M.PvuII (G. M. Adams & R. M. Blumenthal,unpublished results). In contrast, a single-domainstructure has been determined for catechol O-meth-yltransferase (COMtase: Vidgren et al., 1994).Catechol, like cytosine, is a six-membered ring; thissmall molecule can readily diffuse into the activesite of COMtase for methyltransfer from AdoMet.

The structural comparison of three AdoMet-de-pendent Mtases reveals that the catalytic domains ofthe bilobal proteins M.HhaI and M.TaqI, and theentire single domain of COMtase, all exhibit verysimilar three-dimensional folding (Schluckebieret al., 1995). The recently published structure of the5mC Mtase M.HaeIII (Reinisch et al., 1995) is alsoconsistent with this folding pattern. This similarityincludes the positions of amino acid side-chainsinvolved in either AdoMet binding or catalysis. Inother words, many of the conserved motifs in thecatalytic domain of M.HhaI have structural ho-mologs in the other two Mtases (O’Gara et al., 1995).This suggests that many (if not all) AdoMet-depen-dent Mtases may share a common catalytic domainstructure. If so, this not only allows structuralpredictions for other AdoMet-dependent Mtases,but also provides a framework for attempts tocompare their sequences. Guided by this commoncatalytic domain structure, we performed a multiplesequence alignment of 33 N6mA and 9 N4mCMtases. Our results reveal that the N4mC andN6mA Mtases are more closely related to oneanother and to the 5mC Mtases than was expected.

This work confirms that the amino Mtases belongto three groups distinguished by differences in the

Figure 1. Sequence alignment of 33 N6mA DNA Mtases and 9 N4mC DNA Mtases. A, Group a. B, Group b. C, Groupg. Motifs (I to X) are labeled using the nomenclature of Posfai et al. (1989), and sequences are grouped (a to g) usingthe nomenclature of Wilson (1992). Conserved amino acids are grouped as (E, D, Q, N), (V, L, I, M), (F, Y, W), (G, P,A), (K, R) and (S, T), using standard one-letter abbreviations. Invariant amino acids within a group are shown as whiteletters against a black background, conserved hydrophobic positions are indicated by bold letters on a shadedbackground, and conserved polar or charged positions by bold letters within a box. Lesser degrees of conservation areshown, in decreasing order, by bold and uppercase letters, while non-conserved positions are shown as lowercase letters.A (−) indicates a deletion relative to other sequences. Each of the three groups of Mtases is preceded by a theoreticaltopological drawing. Rectangles (lettered) indicate helices, and arrows (numbered) depict strands. Conserved aminoacids from motifs (I to X) are circled and their positions are inferred from the structural comparison of M.HhaI andM.TaqI (Schluckebier et al., 1995). In addition, the secondary structures of M.TaqI, shown in group g, are indicated bycylinders (helices) and arrows (strands) drawn directly above the amino acids forming them. The amino acid sequencesof M.HhaI (a 5mC Mtase) and a small-molecule COMtase are also provided in group g for comparison. (*) Motif X couldnot be identified for M.PaeR7I (N6mA in group g) using sequence P05103, but was readily found in the sequence usedby Wilson (1992), who reported an alternative start position that increases the size of this Mtase from 531 amino acidresidues to 574. (**) The Mtases FokI and StsI are each double-size Mtases with two active halves (Sugisaki et al., 1989;Kita et al., 1992), and each half was analyzed independently.

Figure 1A

Figure 1B

Figure 1C

DNA Amino-methyltransferases 623

linear orders of conserved motifs in their primarysequences. Together with our observation that theAdoMet and methylatable base binding pocketshave remarkably similar structures, this suggestscatalytic roles for several of the conserved side-chains and has implications for the evolutionaryhistory of these enzymes.

Results

Nine conserved motifs of 5mC Mtases arepresent in amino Mtases

The structural similarity of the active site andAdoMet-binding regions of M.TaqI to those ofM.HhaI suggests that the amino Mtases containhomologs of the conserved motifs found in 5mCMtases. The sequences of N6mA and N4mC DNAMtases were therefore gathered and analyzed asdescribed in Materials and Methods. We were pre-pared, for two reasons, to look for these motifs inlinear orders not seen among the 5mC Mtases. First,others had noted that the two previously identifiedconserved motifs in amino Mtases appeared indifferent orders in the various Mtases (Klimas-auskas et al., 1989; Wilson & Murray, 1991; Wilson,1992). Second, others had shown that the 5mCMtases could function with a circularly permutedmotif order (J. Bitinaite, personal communication),or when the regions were expressed separately andallowed to associate in vivo (Karreman & de Waard,1990; Posfai et al., 1991; Lee et al., 1995). We wereable to identify nine segments of sequencesimilarity among the 42 amino Mtases (Figure 1),corresponding to motifs I to VIII and X in the 5mCMtases (Posfai et al., 1989). We could not identify ahomolog to motif IX of the 5mC Mtases; in M.HhaI,this motif is involved in the protein folding of theDNA-recognition region (Cheng et al., 1993a).

In the structures of M.HhaI and M.TaqI, motifs Ito III and X are primarily responsible for bindingAdoMet (Cheng et al., 1993a,b; Klimasauskas et al.,1994; Labahn et al., 1994; Schluckebier et al., 1995),and we term them, collectively, the AdoMet-bindingregion. The structural comparison suggested thatmotifs IV, VI, and VIII are primarily responsible forcatalysis (Schluckebier et al., 1995), as they form theactive site along with motifs V and VII, and we termthem collectively the catalytic region.

Three groups of amino Mtases based onmotif order

Figure 1 clearly shows that the N6mA Mtasescluster into three distinct groups, based on the orderof conserved motifs. It is noteworthy that the N4mCand N6mA Mtases do not group separately fromone another. This grouping is compared to earlieranalyses in the Discussion.

The validity of the grouping shown in Figure 1,which is based solely on motif order, is supportedby the fact that the Mtases within each group aresimilar to one another by several other criteria as

well. First, the groups differ in terms of type ofmethylation (Figure 2A): N6mA Mtases are found inall three groups, but group b includes eight of thenine N4mC Mtases analyzed, and group g has amotif order very similar to that seen in the group ofall 44 sequenced 5mC Mtases (differing only in theposition of motif X; Kumar et al., 1994). Second,comparable motif sequences within each group aremore similar to one another than to the same motifsfrom Mtases in one of the other groups. Third, thegroups differ in terms of molecular mass, withgroup a Mtases being small (260 to 334 amino acidresidues), group g Mtases being large (325 to 580amino acid residues), and group b covering both ofthese size ranges (228 to 531 amino acid residues).Fourth, the groups also differ in terms of the DNAsequence recognized. That is, a distinct consensuscan be derived for each group: while each groupincludes specificities that do not match theconsensus, and some Mtase specificities could fitmore than one consensus, it is clear that the recog-nition specificities are non-randomly distributedamong the groups. As indicated in Figure 1, 12/12group a N6mA Mtases recognize the sequence(C/G)MN(0-2)T(G/C) (M = A/C; underlining indi-cates the methylated base), 14/17 group b Mtasesrecognize the sequence (G/C)N(0-3)MN(0-2)(G/C),and 11/12 group g Mtases recognize the sequenceTNNA (the one exception is M.EcoRI). Theseconsensus substrate specificities allow verifiablepredictions. For example, the nucleotide sequence ofthe ClaI Mtase gene has not been reported, but itsspecificity (ATCGAT) suggests that it will be foundto belong to group g.

The Mtases differ in the relative linear order ofthree regions: the AdoMet-binding region, thecatalytic (active site) region, and the targetrecognition region (Figure 2). In the 5mC Mtases,the target recognition region is responsible forspecific DNA sequence recognition, and is generallylocated within the longest gap between conservedmotifs (Klimasauskas et al., 1991; Mi & Roberts,1992; Noyer-Weidner & Trautner, 1993). Group a isarranged in the order (amino to carboxy): AdoMet-binding region, target recognition region, and thencatalytic region. Group b is arranged in the order:catalytic region, target recognition region, andAdoMet-binding region. Group g is arranged in theorder: AdoMet-binding region, catalytic region, andtarget recognition region. No Mtases were found tohave the predicted AdoMet binding region betweenthe other two regions (Figure 2A, arrangements dand z). No Mtase is known to have the targetrecognition region at the amino end (Figure 2A,arrangements e and z); however, M.VspI (N6mA,assigned to group g), M.CfrBI and M.BamHI (bothN4mC, assigned to group b) have long (>100 aminoacid residue) amino-proximal sequences upstreamof the first conserved motif, and in theory theseupstream sequences could contain the targetrecognition regions for those three Mtases. If so,M.VspI could be assigned to group z and M.CfrBIand M.BamHI to group e.

DNA Amino-methyltransferases624

A

B

Figure 2. A, Possible arrangements of the three major regions found in DNA Mtases. To the right, the representationof these arrangements is indicated for Mtases that generate 5mC, N6mA, or N4mC. The asterisk (*) refers to the factthat 5mC Mtases have one major difference from the group g amino Mtases: motif X is near the carboxy terminus in5mC Mtases. B, Ten representative examples of 5mC, N6mA, and N4mC Mtases, aligned by motif IV and showing therelative positions of the other conserved motifs. The longest variable region is indicated as the putative target recognitionregion (labeled as the TRD) in each case.

DNA Amino-methyltransferases 625

A B

C D

Figure 3. Superimposition of the two a/b clusters from DNA Mtases, in ribbon representation (Carson, 1991). Thesuggested duplication is not obvious from examination of the primary sequences of M.HhaI or M.TaqI (but see Laurentset al., 1994). A, B, M.HhaI. The two a/b clusters b1 : aA : b2 : aB (shown in green with G-loop in yellow)and b4 : aD : b5 : aE (shown in brown with P-loop in cyan) were isolated from the co-crystal-derivedM.HhaI–DNA–AdoHcy structure (A), and rotated with respect to one another to achieve the most overlapping possible(B). The b-sheets from the two a/b clusters could be superimposed with an r.m.s.d. of <1 A for the Ca atoms. Alsoshown are the positions, relative to the respective a/b clusters, of the AdoHcy adenosyl moiety (green) and the targetcytosine ring (brown). C, D, M.TaqI. Presented as described for M.HhaI, except that the target adenine ring is not shown,since its structural position has not yet been determined.

Comparison of conserved motifs among theMtase families

While the order of conserved motifs variesamong the three groups of Mtases, many oftheir basic features are, by definition, re-tained. These conserved features are describedbelow.

Motif I

This motif has been described in a variety ofAdoMet-dependent Mtases (see Introduction).Structurally, motif I forms the secondary structureb1-loop-aA (Figure 1; Schluckebier et al., 1995). Gly,and less frequently Ala or Pro, form the loop(G-loop) that binds the methionine moiety of

DNA Amino-methyltransferases626

AdoMet. The core of the G-loop is the Gly-X-Glytripeptide (X is any amino acid), but three group gMtases in Figure 1 replace the first Gly with Ala orSer (M.EcoRI), while nine (six of them in group a)replace the second Gly with one of sevenalternatives. The majority of amino Mtases havePro as the last amino acid of strand b1 (Pro46 inM.TaqI), but seven of 13 Mtases in group a (andthe 5mC Mtases) have Ile or Leu at that position(Leu17 in M.HhaI). Conserved hydrophobic side-chains in strand b1 are required for packingagainst helix aA. The only motif I rule withoutexceptions among the Mtases in Figure 1 involvesthe position 4 amino acids upstream of theGly-X-Gly, which is, in all cases, Asp or Glu. Thisis the penultimate position of b1 (Figure 1), andposes additional stereochemical constraints byinteracting with the dipoles of the peptide bonds inthe G-loop.

The most pronounced motif I difference amongthe Mtases is that both groups a and b, as well asthe 5mC Mtases, have Phe at the beginning of theG-loop (second position amino to Gly-X-Gly), butgroup g Mtases have Ala, Ser or Gly instead: justtwo Mtases in Figure 1 violate this pattern (M.EcoRIin group g and M.HhaII in group b). In thestructure of the 5mC Mtase M.HhaI, this G-loopPhe18 forms an edge-to-face van der Waals (vdw)contact with the adenine moiety of AdoMet.However, in the structure of M.TaqI (a member ofgroup g), the same interaction with AdoMet isprovided by the Phe146 ring from helix aD(Schluckebier et al., 1995). It appears that, in groupg, the Phe that begins the G-loop in the 5mC Mtasesis replaced by a spatially equivalent Phe from helixaD/motif V (see below).

Motifs II and III

These two motifs were described as less-con-served blocks in 5mC Mtases (Posfai et al., 1989). Inthe structurally characterized Mtases, motif IIcontains a negatively charged amino acid at the lastposition in strand b2, interacting with the ribosehydroxyls of AdoMet, and followed by a bulkyhydrophobic side-chain that makes vdw contactswith the AdoMet adenine (Glu40-Trp in M.HhaI,Glu71-Ile in M.TaqI). Of the 42 amino Mtases inFigure 1, 30 match a (Glu/Asp)-F consensus, whereF is any bulky hydrophobic side-chain, usuallyfollowed by Asp, Glu or Asn. The groups do notdiffer substantially in this.

Motif III also contains an Asp/Glu or Asn/Gln inthe first position of aC (Asp60 in M.HhaI and Asp89in M.TaqI), which interacts directly with theexocyclic NH2 (N6) of the AdoMet adenine(Schluckebier et al., 1995). Motif III, in addition,provides a hydrogen bond to N1 of the AdoMetadenine from a peptide backbone NH group (Ile61in M.HhaI and Phe90 in M.TaqI). The correspondingposition is group-specifically conserved in theamino Mtases (Figure 1).

Motif IV

What we call motif IV of the amino Mtases wasfound in early sequence comparisons and called a‘‘DPPY motif’’ based on its sequence (Hattmanet al., 1985; Chandrasegaran & Smith, 1988). A latercomparison of 16 N6mA and three N4mC Mtasesidentified only two conserved segments (Kli-masauskas et al., 1989), one of which is motif I. Theother conserved segment was the DPPY motifwhich, it was suggested, might correspond to motifIV in 5mC Mtases, even though the reactionmechanisms appear to be quite distinct. Thestructural comparison of M.HhaI and M.TaqI haveconfirmed this correspondence (Schluckebier et al.,1995). This diprolyl motif is located in the loopregion outside the carboxyl end of b4 (the P-loop;Figure 1). The P-loop forms the active site, alongwith motifs VI and VIII (see Discussion). Thepeptide backbone of the corresponding P-loop inM.HhaI also contributes to the AdoMet binding site(Cheng, 1995a; see also Kossykh et al., 1993). MotifIV has the consensus sequence Asp-Pro-Pro-Tyr(DPPY) in group a, DPPY for N6mA Mtases ingroup b, Asn-Pro-Pro-Tyr (NPPY) in group g, andSer-Pro-Pro-Tyr (SPPY) for N4mC Mtases; thisgrouping pattern for motif IV has been notedpreviously (Wilson & Murray, 1991; Wilson, 1992).There are exceptions to this pattern, most notablyM.BamHI (N4mC in group b, which has a DPPF),M.HhaII (N6mA in group b, DPQY) and M.StsI-a(N6mA in group a, DTPY).

Motif V

In group g, motif V contains the consensus(Asn/Asp)-Leu-Tyr-X-X-Phe-(Leu/Val/Ile). As de-scribed above, in group g, this Phe replaces the Phethat begins the G-loop in the Mtases of groups a orb. The Leu (Leu100 in M.HhaI, Leu142 in M.TaqI)makes vdw contacts to the AdoMet adenine on thesame side as the Phe (Schluckebier et al., 1995). Ofthe Mtases in Figure 1, only one that lacks Phe at thestart of the G-loop fails to contain it in motif V, andthat is M.HhaII, which has a Ile at that point. Ingroups a and b, one of the conserved hydrophobicside-chains in motif V may have the same spatialposition as the Leu in group g Mtases.

Motifs VI, VII and VIII

In the structurally characterized Mtases, motif VIforms strand b5 (Figure 1; Schluckebier et al., 1995).A conserved Gly starts the strand, and the strandends with Gly, Pro or Ala, while in group a itends with Ser-Asn. Groups g and b also differfrom group a in having a conserved pattern ofhydrophobic amino acids in b5.

Motif VII is not strongly conserved even among5mC Mtases, yet credible candidates can be foundwithin each group. In M.HhaI, this motif includesAsp144-Tyr in the loop between helix aE and strandb6, and it faces away from the DNA-binding cleft

DNA Amino-methyltransferases 627

(Cheng et al., 1993a). It is thus believed to beinvolved in the folding of the catalytic region(Cheng, 1995b).

In the primary sequence, motif VIII bears littleresemblance to the motif present in 5mC Mtases(Gln161-X-Arg-X-Arg165 in M.HhaI). This pre-sumably reflects the fact that the 5mC Mtasesinteract with cytosine via hydrogen bonds (throughArg165 in M.HhaI), while the N6mA Mtases appearto interact with the target DNA adenine viahydrophobic interactions. In the structure of M.TaqI,the corresponding region (the loop connectingstrands b6 and b7) contains Phe196, which aligns toa conserved Phe or Tyr in other amino Mtases. It issuggested that Phe196 makes favorable edge-to-faceor face-to-face vdw contacts to the target DNAadenine (Schluckebier et al., 1995).

Motif X

The location of motif X in the primary sequenceis one of the major differences between the 5mCand amino Mtases. In the 5mC Mtases, this motifcomes from the carboxy terminus. In the aminoMtases, the corresponding motif is always to theamino side of motif I: at the amino terminus of theprotein in groups a and g, and in the middle of theprotein in group b. There are pronounced group-specific differences in the sequence of this motif(Figure 1). However, in all Mtases, this motif isexpected to form a helix next to strand b1 (formedby motif I), with conserved hydrophobic side-chainsrequired at certain positions for packing against theb-strands, and a loop preceding the helix (Figure 1).This loop, along with the G-loop of motif I and theP-loop of motif IV, form the sides of the bindingpocket in M.HhaI for the methionine moiety ofAdoMet (Cheng, 1995a).

Discussion

Structural comparison of the Mtase groups

The identification of nine conserved motifs sharedwith the 5mC Mtases allows the amino Mtases fromeach group to be mapped onto the consensusstructure in a systematic manner (Figure 1). Themost pronounced difference among these threegroups of amino Mtases is the connection betweenthe proposed AdoMet-binding and catalytic re-gions. In group g, a connection between helix aCand strand b4 links the two regions; M.TaqI belongsto this group. M.HhaI and COMtase also belong togroup g, based on the order of the conserved motifs(Figure 1; excepting motif X in the case of the 5mCMtase M.HhaI), meaning that all currently availableMtase structural information is from Mtases with,essentially, a group g motif order. In groups a andb, the two regions are apparently connected via aseparate domain, the target recognition region. Thecatalytic and AdoMet-binding regions of theseMtases could nevertheless fit the consensus

M.HhaI–M.TaqI structure. Whether this actuallyoccurs is currently being explored, as several moreDNA Mtases are undergoing crystallographicanalysis. As the group a and b Mtases are proposedto have the DNA recognition domain between theAdoMet-binding and catalytic regions, it is interest-ing that other structurally characterized proteinshave a recognition domain inserted betweensequences that form a catalytic b-sheet cluster (forexample, the G protein, Coleman et al., 1994; theR.PvuII endonuclease, Cheng et al., 1994), and thattwo flavoproteins have different domains insertedbetween parts of the FAD-binding domain (Mittl &Schulz, 1994; Schreuder et al., 1994).

Catalysis of N6-adenine methylation

What can the consensus M.HhaI–M.TaqI structureand the conservation of nine sequence motifs amongthe amino and 5mC Mtases tell us about thepossible catalytic mechanism of the N6mA Mtases?We propose that the answer to this question lies ina comparison of the binding sites for DNA adenineand for the adenosyl moiety of AdoMet, which arestrikingly similar. While no N6mA Mtase–DNAco-crystal structure has yet been determined, theconservation of structure and function amongAdoMet-dependent enzymes is supported by boththe similar structural framework of the catalyticdomains found in M.HhaI, M.TaqI, and COMtase,and by the similar conformation of the boundAdoMet with the methyl group positioned (notsurprisingly) close to the substrate (Schluckebieret al., 1995). This structure-function conservation isalso suggested by the conservation of amino acidsfrom motifs I, II, III, V, and X which, in thestructurally characterized Mtases, interact withAdoMet.

Are the DNA–adenosyl and AdoMet–adenosylbinding sites structurally comparable? They areeach dominated by comparable a/b clusters(b1 : aA : b2 : aB and b4 : aD : b5 : aE):the former includes motifs I and II, and forms thebulk of the AdoMet-binding region, and the latterincludes motifs IV to VI, and forms the bulk of thecatalytic region. These two a/b clusters and theirbound substrates do, in fact, have strikingly similarstructures. The two a/b clusters from the M.HhaI–DNA–S-adenosyl-L-homocysteine (AdoHcy) struc-ture can be superimposed, with a root-mean-squaredeviation (r.m.s.d.) of <1 A for the Ca atoms in theb strands, with the AdoHcy adenosyl moietyoverlapping the target cytosine ring (Figure 3Aand B). Similar overlapping is also possible for thea/b clusters of the M.TaqI–AdoMet structure(Figure 3C and D) and of the M.HaeIII–DNAstructure (results not shown). While the variousMtase groups have these two a/b clusters indifferent orders (Figures 1 and 2), in no case has themotif order rearrangement interrupted an a/bcluster (Figure 1). The relatedness of the bindingpockets for the DNA base and for AdoMet may

DNA Amino-methyltransferases628

explain an interesting feature of the M.HaeIIIstructure (Reinisch et al., 1995): the unpaired5' thymine of one DNA duplex penetrates theAdoMet pocket of the neighboring Mtase–DNAcomplex. The thymine does not enter deeply enoughto interact with the conserved acidic amino acidside-chains (see section on motifs II and III), butdoes make the hydrophobic contacts made by theAdoMet adenosyl moiety, such as face-to-facestacking with Tyr30 of motif II (analogous to Trp41in M.HhaI and Ile72 in M.TaqI).

Based on the chemical and structural similarity ofthe DNA–adenosyl and AdoMet–adenosyl moieties,and the structural similarity of the AdoMet-bindingand catalytic regions of the Mtase, we proposeanalogous Mtase–adenosine interactions in the tworegions (Table 1). The methylation of adenineappears to result from a direct attack of the AdoMetmethyl group on the adenine N6 (Pogolotti et al.,1988; Ho et al., 1991).

In analogy to the hydrogen bond betweenAdoMet–adenosyl N6 and a motif III Asp in M.HhaIand M.TaqI, we suggest that the N6 amino nitrogenof the target adenine is the donor in a hydrogenbond to the side-chain of Asp/Asn in motif IV, andpossibly to one of the main-chain oxygens of theadjacent two proline residues. This would nega-tively polarize N6, activating it for direct transfer ofthe CH+

3 from AdoMet. In Mtases with Asn in thisposition (group g) the carboxamide could be thedonor in a hydrogen bond to adenine N1, as well asan acceptor from adenine N6, similar to the roleAsn229 of thymidylate synthase plays in hydrogenbonding to dUMP (Liu & Santi, 1993; also see Figure3 of Gerlt, 1994). Mtases with Asp in this positioncould also hydrogen bond adenine N1 if thecarboxyl is protonated.

Consistent with the above, mutation of DPPY toGPPY or APPY in the two halves of the bifunctional

Mtase M.FokI (group a) abolishes activity in thealtered half (Sugisaki et al., 1989). Altering DPPY toSPPY or NPPY abolishes the activity of the group aN6mA Mtase M.EcoDam (Guyot et al., 1993). TheM.EcoDam result is somewhat surprising, as NPPYis in motif IV in nine of 12 Mtases of group g(Figure 1), while SPPY is in motif IV in six Mtasesof group b and even one (M.MvaI) of group a (seethe following section on N4mC Mtases). Similarly,altering NPPF to DPPF in M.EcoKI led to loss ofactivity (Willcock et al., 1994). (M.EcoKI is a type IN6mA Mtase that has also been modeled onto theconsensus M.HhaI–M.TaqI structure (Dryden, et al.,1995) We interpret these data from mutant enzymesto mean that the relative positions of the activatinghydrogen bond acceptor, target amino group, andAdoMet methyl group must be precisely main-tained.

The two proline residues are not present in allexamples of motif IV: M.HhaII (group b; DPQY) andM.StsI-a (group a; DTPY) resemble the rRNAN6mA Mtases in this respect. Analysis of 12 rRNAN6mA Mtases (EC 2.1.1.48) reveals the consensus(N/S)IP(Y/F) (X. Cheng, unpublished obser-vations). Altering motif IV of the bacteriophage T4Dam N6mA Mtase from DPPY to DAPY or DTPY(as occurs in M.StsI-a) substantially increasedKAdoMet

M , but had much smaller effects on kcat, KAdoMeta

and KDNAM (Kossykh et al., 1993). This suggests that

the Pro alteration affects a catalytic but not arate-limiting (not kcat-determining) step, consistentwith the above inferences (particularly if productrelease is the rate-limiting step, as it is for at leastsome Mtases: Reich & Mashoon, 1993). Unfortu-nately, these mutations have not been made inM.EcoDam, but in that enzyme, changing DPPY toDGPY, DVPY, DPGY, DPRY, DPQY (as occurs inM.HhaII), DPEY, or DPVY all abolished activity(Guyot et al., 1993). In summary, these results are

Table 1. Comparison of the DNA–adenosyl and AdoMet–adenosyl binding sites in group ga

Target adenine ring AdoMet adenine ring Possible functions

Motif aa Location Motif aa Location

(A) IV Asn First aa of P-loop III Asp/Ser First aa of aC Side-chain hydrogen bonds toN6-nitrogen.b

(B) c c c III Phe/Tyr Second aa of aC Main-chain NH group hydrogenbonds to N1-nitrogen.

(C) IV Tyr/Phe/Trp Forth aa of P-loop Vd Phed aDd Edge-to-face vdw contact withadenine.

(D) VI Ile/Val Penultimate aa of b5 V Leu/Ile/Tyr Last aa of P-loop vdw contact with adenine ringon the same face as in (C).

(E) VIII Phe Loop between strands II Ile/Val/Leu/Phe First aa of loop Face-to-face vdw contactb6 and b7 between strand b2 with adenine ring on the

and helix aB opposite face.a See also Figure 1C.b N6 of target adenine could form a second hydrogen bond to one of the main-chain oxygen atoms of the two proline residues in

motif IV at the P-loop.c The amide side-chain of Asn in (A) could be a hydrogen bond donor to N1 of adenine. If the carboxyl of Asp is protonated, it

could also be hydrogen bond donor to adenine N1 (N6mA Mtases in groups a and b) or to cytosine N3 (N4mC Mtase M. BamHI).For N4mC Mtases with Ser at the first position of the P-loop, a conserved Asn from motif VI (in the end of strand b5) could possiblyhydrogen bond cytosine N3 in analogy to Glu119 in motif VI of M.HhaI.

d In groups a and b, the phenyl ring is from motif I, the first amino acid (aa) of the G-loop.

DNA Amino-methyltransferases 629

consistent with the role suggested for motif IV, butalso make clear the dangers of considering themotifs in isolation.

The Tyr in motif IV, Phe in motif VIII, andhydrophobic side-chains in motif VI could functionin properly orienting the target DNA adenine. Theanalogy for this Tyr:Phe pair is the Phe from motifV (group g) or motif I (groups a and b) that makesan edge-to-face vdw contact with the AdoMetadenine (Table 1), and to the Trp41 of motif II inM.HhaI, which makes a face-to-face vdw contactwith the AdoMet adenine. This interpretation isconsistent with the fact that altering motif IV of theM.EcoKI N6mA Mtase from NPPF to NPPG orNPPC abolished activity without greatly affectingthe affinity for DNA or AdoMet (Willcock et al.,1994). In contrast, altering the NPPF to NPPY orNPPW (as occurs in M.VspI) resulted in an enzymethat retained partial activity (Willcock et al., 1994).

Catalysis of N4-cytosine methylation

From the first report of an N4mC Mtasesequence, it has been suggested, based on overallsequence similarity and the very similar chemicalproperties of adenine N6 and cytosine N4 (e.g. seeFigure 3 of Weiner et al., 1984; Renugopalakrishnanet al., 1971), that N4mC and N6mA Mtases may usea common reaction mechanism (Tao et al., 1989). Webelieve that N4-cytosine and N6-adenine methyl-ation do use the same catalytic mechanism, for thefollowing reasons. First, as described above, theN6mA and N4mC Mtases in group b appear to bemore closely related to one another than eithersubgroup is to the N6mA Mtases of group g.Second, the N6mA and N4mC versions of motifs areeither indistinguishable from one another, or are notconsistently different from one another (the excep-tions are usually provided by M.MvaI or M.BamHI).For example, the most obvious difference betweenthe N4mC and N6mA Mtase sequences is theconserved Ser present in motif IV in place ofAsp/Asn; yet this Ser must not represent anessential functional difference, as it is not present inthe N4mC Mtase M.BamHI. We suggest that the Serin motif IV of most N4mC Mtases could hydrogenbond and activate the amino N4 nitrogen of thecytosine ring, in analogy to COMtase, in which a Seris hydrogen-bonded to the AdoMet N6 (Vidgrenet al., 1994), and in analogy to the Asp in motif IIIof the N6mA Mtases (Table 1). These Mtases mayalso be hydrogen bond donors to cytosine N3,similar to the proposed bonding of N6mA Mtasesto adenine N1, but the donor side-chain would befrom outside motif IV (possibly a conserved Asn inmotif VI). Other requirements for N4 methylation ofcytosine may be implied by the fact that theindividual motifs in M.MvaI, while unambiguouslyin the group a order, more closely resemble thecorresponding motifs of the other (group b) N4mCMtases (Figure 1).

DNA Mtase families and comparison to earlierMtase groupings

It should be noted that the results of our analysisare very consistent with, and provide a structuralbasis for, earlier attempts to group Mtases. The firstof these attempts examined 17 Mtases, and wasbased not on motif identification and order but onoverall sequence alignment (Chandrasegaran &Smith, 1988). That analysis found five groups ofMtases. Their group I included four Mtases all inour group a; their group II included two Mtasesboth in group b; their group III included fourMtases all in group g; their group IV included six5mC MTases (which we did not include, but whichhave a group g motif order, except for the positionof motif X); and their group V consisted of M.EcoRI,which has several variations from the consensusmotifs, but which we assign to group g based onmotif order. The second major analysis categorized33 type II amino Mtases (Wilson & Murray, 1991).They placed the amino Mtases into five groups,based on the order and nature of just motifs I andIV (again, M.EcoRI was not grouped). Their analysisis consistent with our assignments except that (1)they grouped the N4mC and N6mA Mtasesseparately, and (2) they assigned M.SmaI differently.The N4mC and M6mA Mtases were not separatedin a later version of that analysis (Wilson, 1992), andwe have adopted the nomenclature of that lateranalysis. Four to ten Mtases from group g were alsoclustered by Lauster et al. (1987), Janulaitis et al.(1992), and Noyer-Weidner et al. (1994), based onoverall sequence similarity. More recently, Timin-skas et al. (1995), using a sensitive method to makepairwise comparisons between amino Mtases,detected two conserved motifs in addition to thetwo that had been identified (see Klimasauskaset al., 1989). Their conserved motif (CM) Is cor-responds to motif X, CM I to motif I, CM II to motifIV, and CM III to motifs V and VI. Using these, theyhave defined eight groupings for the Mtases whichare consistent with ours and with Wilson & Murray(1991); Timinskas et al. (1995) have just subdividedtheir groupings to separate N4mC and N6mAMtases. Since our analysis began with assignmentof motifs I and IV (Materials and Methods), it is notsurprising that we got our grouping consistent withWilson & Murray (1991), but the discovery of theseven additional conserved motifs in consistentorders greatly strengthens the basis for this Mtasecategorization.

Evolutionary implications

The four DNA Mtase arrangements seen to date(a, b, g, and 5mC) differ in the linear order ofconserved motifs (Figure 2), but in no case areeither of the two a/b clusters interrupted. Further-more, consider the superimposable structures ofthose clusters, which appear to have the same linearorder in all four groups (b1/b4 : aA/aD : b2/b5 : aB/aE; Figure 3), and the apparent functional

DNA Amino-methyltransferases630

relatedness of the AdoMet and adenine bindingsites (Table 1). All of this is consistent with thepossibility that the original Mtases arose after geneduplication converted an AdoMet-binding proteininto a protein that bound two molecules of AdoMet(see also Lauster, 1988, 1989; Tao et al., 1989;Guyot & Caudron, 1994). The two halves couldhave diverged when either the amino-proximal orcarboxy-proximal AdoMet-binding site evolved tobind adenine, and then diverged further to yieldCOMtase (Figure 1) and the wide variety of otherAdoMet-dependent Mtases (Fujioka, 1992; Clarke,1993). To become DNA Mtases, by this duplicationmodel, an additional fusion would have brought thetarget recognition region in at the carboxy terminus(group g) or between the AdoMet and adeninebinding sites (groups a and b) of the ancestraladenine Mtase. This duplication model may provideinsight into the fact that some Mtases appear to bindtwo molecules of AdoMet (Bergerat & Guschlbauer,1990; Adams & Blumenthal, 1995), one of whichaffects the selectivity between substrate andnon-specific DNA sequences (for M.EcoDam;Bergerat & Guschlbauer, 1990). It is also noteworthy,with regard to the proposed importation of a targetrecognition region, that some 5mC Mtases arenaturally made as two separate polypeptides: onehas motifs I to VIII (including both a/b clusters)and the other carries the target-recognizing regionand motifs IX and X, and these associate in the cellto form active enzyme (Karreman & de Waard, 1990;Lee et al., 1995).

While the 5mC and N4mC Mtases each fall intosingle groups defined by motif order (with oneexception, M.MvaI; N4mC Mtase assigned to groupa), the N6mA Mtases we examined are fairly evenlydistributed among the three groups (27% in groupb, and just over 36% each in groups a and g). Thismay also be explained by a model in which theoriginal nucleic acid Mtase(s) generated N6mA.

In summary, the DNA Mtases appear to be,paradoxically, both more uniform (shared conservedmotifs; N4mC Mtases not a distinct group) andmore diverse (four possible motif orders) than hadbeen expected. The solved structures of Mtasesfrom groups a and b should be very informative.

Materials and MethodsThe Swissprot database was searched by EC number,

yielding the amino acid sequences of 33 N6mA DNAMtases (EC 2.1.1.72) and nine N4mC DNA Mtases (EC2.1.1.113). The names and accession numbers of theseMtases are listed in Figure 1. For comparison, we alsoused the sequences of the 5mC Mtase (EC 2.1.1.73)M.HhaI and the small-molecule COMtase (EC 2.1.1.6).

A scan of the sequences was first performed to locatemotif I and motif IV. These two blocks provided theanchor points for global alignment of other motifs. Ascomparable motifs did not always appear in the samelinear order, the alignments were refined within eachMtase group. Our analysis of M.EcoRI yielded a differentmotif I assignment from that of Klimasauskas et al. (1989).For clarity and convenience, we retain the nomenclature

of Posfai et al. (1989) for the 5mC Mtase conserved motifsand of Wilson (1992) for the Mtase groups. TheM.HhaI–M.TaqI structural alignment was crucial to thisanalysis, as it indicated which motif positions were mostfunctionally significant and which substitutions werelikely to be permissible.

AcknowledgementsWe thank Catherine Luschinsky Drennan (University of

Michigan) for critical discussion of the structuralimplications, Markus Winter and David T. F. Dryden(University of Edinburgh) for discussion of the groupassignments, Gerd Schluckebier and Wolfram Saenger(Freie Universitat) for the coordinates of M.TaqI, andKarin M. Reinisch and William N. Lipscomb (HarvardUniversity) for the coordinates of M.HaeIII. We also thankAshok S. Bhagwat and Joan C. Dunbar (Wayne StateUniversity), David T. F. Dryden (University of Edin-burgh), Stanley Hattman (University of Rochester),Rowena G. Matthews and Joseph T. Jarrett (University ofMichigan), and Richard J. Roberts, Sanjay Kumar, JanosPosfai, and Geoffrey G. Wilson (New England Biolabs) forhelpful discussions and comments on the manuscript.This work was supported by the W. M. Keck Foundation(to X.C.), by grant GM49245 from the National Institutesof Health (to X.C.), and by grant DMB-9205248 from theNational Science Foundation (to R.B.).

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Edited by D. E. Draper

(Received 14 June 1995; accepted 11 August 1995)