In previous studies it was demonstrated that the only DNA MTase in E. histolytica
, EhMeth, belongs to the DNMT2-family and has dual substrate specificity being able to methylate DNA as well as C38 of tRNAAsp
. The three-dimensional structure of EhMeth shows a high structural homology to the bacterial DNA MTase M.Hha
I and to human DNMT2, which could make the design of a species-specific inhibitor a challenging task. The structural homology manifests itself not only in the general fold, but is also present in all elements required for methyl-group transfer and in many characteristics required for DNA binding ( & S1). This homology suggests that EhMeth utilizes the same reaction mechanism for methyl-group transfer as shown for DNMT2 and M.Hha
. The observed AdoHcy conformation as well as the DNA-binding model presented in this study, further supports this hypothesis.
However, the high similarity between bacterial DNA MTases and DNMT2 enzymes raises the question what structural differences exist, that prevents the bacterial enzymes from methylating tRNA and allows DNMT2 enzymes to select only for specific tRNAs.
While the large domain, required for methyl-group transfer, is relatively invariant among MTases, the small domain has been associated with altered substrate specificity 
. In line with this the small domains of M.Hha
I, DNMT2 and EhMeth display indeed notable differences to each other. While all small domains of the three MTases share the short, central β-propeller motif, the surrounding elements relevant for substrate recognition are different. The small domain of M.Hha
I is almost exclusively β-stranded, whereas the small domain in DNMT2 and EhMeth is predominantly α-helical. In spite of this difference, a single residue located in strand β10 in M.Hha
I that was shown to interact with DNA, is also conserved in DNMT2 and EhMeth 
. However, the residues forming strand β10 in M.Hha
I are shifted more to the proximal side in DNMT2 and EhMeth and the conserved residue (Arg226 in EhMeth) cannot be found at a structurally equivalent position ( & S2). Notably the relocation of strand β10 is accompanied with the formation of an acidic pocket at a Φ-D-I-V motif (where Φ is a hydrophobic residue) that is strictly conserved in DNMT2-MTases ( & S1). Size and shape of the acidic pocket suggest that it has ideal proportions to accommodate the base of a nucleotide, thus could play a role in substrate binding. At first glance this may appear to be a subtle difference but due to the strong conservation this clearly discriminates DNMT2 enzymes from bacterial MTases. Furthermore, this conserved change is one of the few highly charged positions on the distal side of EhMeth ( & S2), and in direct proximity to an area that was shown to be important for substrate binding in M.Hha
I. Furthermore, no substantial difference between EhMeth and the bacterial MTase can be observed at the proximal side of the enzymes ().
One explanation for the conservation of the acidic pocket could be an altered sequence specificity of DNMT2 enzymes. EhMeth was shown to methylate specific sequences in S/MAR regions and LINE retrotransposons 
. This demonstrates target selectivity among DNA sequences, and indicates a role of EhMeth in the regulation of retrotransposons among Entamoeba
species. However, the conservation of the acidic pocket could also be explained by the ability of DNMT2 enzymes to recognize specific tRNAs. In support of the latter hypothesis, the conserved Arg226 as well as the Φ-D-I-V motif are in close proximity to the target recognition domain (TRD) harboring the CFT sequence motif (CFTxxYxxY, where x is any amino acid), which is unique to DNMT2 enzymes 
To decipher the function of DNMT2-MTases, another important question is to understand what distinguishes the target tRNAs from non-cognate tRNAs. Crystal structures of tRNA-modifying enzymes bound to tRNA reveal that tRNA recognition often not only depends on the nucleotide to be modified, but also requires interactions with additional parts of the tRNA 
. For the bacterial tRNA-MTase TrmA, it was suggested that in addition to the T-loop the D-loop interacts with the protein 
. Similarly, the eukaryotic tRNA-MTase Trm5 interacts not only with the anticodon loop harboring the substrate nucleotide G37, but also contacts the D- and T-loop 
. Both loops have been reported to be more stable in fully modified and mature tRNA molecules 
. As a consequence, Trm5 specifically selects and modifies tRNA species, which adopt the correct conformation and thus may also serve as a sensor for proper folding and accurate tRNA maturation 
. TiaS in contrast recognizes the acceptor stem of the tRNA in addition to specific interactions with the substrate nucleotide C34 located in the anticodon loop 
Clearly, DNMT2 enzymes such as EhMeth do not discriminate against the anticodon sequence of the tRNA itself, since the enzyme was shown to methylate tRNAAsp
as well as tRNAVal
. A singular discriminating feature is also unlikely to be found in the anticodon stem loop, since the mobility shift assays clearly show that EhMeth depends on additional parts of the L-shaped tRNA to recognize its substrate in a stable manner (). Therefore, the discriminating property of the target tRNAs has to be found in other areas of the tRNAs. Indeed, alignments of target tRNAAsp
from different organisms reveal a common sequence motif (UAGUNΨ) located at the 5′ end of the D-stem (Figure S4
). Hence, it is tempting to speculate that the D-stem plays an important role in substrate recognition. However, also tRNAHis
comprise this motif in their D-stem indicating that additional criteria need to be fulfilled for substrate discrimination. Most obvious is the presence of a guanosine at position 39 in tRNAAsp
. Interestingly, these three tRNAs share a cytosine at the third position of the anticodon sequence.
To understand the dual substrate acceptance of DNMT2 MTases and the specificity for three tRNAs one inevitably needs to analyze a DNMT2-tRNA complex structure. The co-crystal structure of the methyltransferase with its substrate tRNA will provide profound insights into the protein-tRNA interactions involved in this fundamental process.
The crystal structure of the cytosine-5-methyltransferase EhMeth (DNMT2) in complex with AdoHcy shows high structural similarity to the DNA MTase M.HhaI and to human DNMT2. Mobility shift assays show the requirement of the full-length tRNA for stable complex formation in vitro. We hypothesize that a conserved region on the distal side of EhMeth could be involved in tRNA binding.