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The cofactor-binding pocket. (A) A surface representation of EhMeth shows the cofactor-binding pocket in the large domain of the protein. The binding of AdoHcy in the pocket is mediated by a variety of polar and non-polar interactions shown in the close-up view. A |FO−FC| simulated annealing omit map contoured at 1.5 σ (blue mesh) supports the conformation of the ligand. AdoHcy is shown in a green while the interacting residues are shown in a grey ball and stick representation. (B) A LIGPLOT diagram shows the 2-dimensional projection of the protein residues interacting with the ligand. Distances between individual interacting atoms are indicated.
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In eukaryotes, DNA methylation is an important epigenetic modification that is generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2 family have been shown to have a dual substrate specificity acting on DNA as well as on three specific tRNAs (tRNA(Asp), tRNA(Val), tRNA(Gly)). Entamoeba histolytica is a major human pathogen...
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Dimers of tRNAs can form through quasi self-complementary anticodon-anticodon interactions, for example at neutral pH in yeast tRNA Asp(GUC) and at pH 4.5 in Escherichia coli tRNA Gly(GCC) through a partially protonated interaction. The requirements for tRNA oligomerization, and the factors that prevented higher orders of structures forming were ex...
Citations
... S-Adenosyl-L-homocysteine (SAH), as a SAM product in the catalysis reaction, only lacks a methyl group. Except for E. histolytica DNMT2/TRDMT1-SAH complex, three complexes in human, Schizosaccharomyces pombe (S. pombe) and Spodoptera frugiperda (S. frugiperda) are crystallized without RNA supplement [23,[34][35][36]. Even so, SAH in each crystal structure is located in an active orientation ( Figure S10 C, D, E, F). ...
RNA methyltransferase DNMT2/TRDMT1 is the most conserved member of the DNMT family from bacteria to plants and mammals. In previous studies, we found some determinants for tRNA recognition of DNMT2/TRDMT1, but the preference mechanism of this enzyme for substrates tRNA and DNA remains to be explored. In the present study, CFT-containing target recognition domain (TRD) and target recognition extension domain (TRED) in DNMT2/TRDMT1 play a crucial role in the substrate DNA and RNA selection during the evolution. Moreover, the classical substrate tRNA for DNMT2/TRDMT1 had a characteristic sequence CUXXCAC in the anticodon loop. Position 35 was occupied by U, making cytosine-38 (C38) twist into the loop, whereas C, G or A was located at position 35, keeping the C38-flipping state. Hence, the substrate preference could be modulated by the easily flipped state of target cytosine in tRNA, as well as TRD and TRED. Additionally, DNMT2/TRDMT1 cancer mutant activity was collectively mediated by five enzymatic characteristics, which might impact gene expressions. Importantly, G155C, G155V and G155S mutations reduced enzymatic activities and showed significant associations with diseases using seven prediction methods. Altogether, these findings will assist in illustrating the substrate preference mechanism of DNMT2/TRDMT1 and provide a promising therapeutic strategy for cancer.
... The protein showed homology and high structural similarity with the human methyltransferase Dnmt2 and can methylate DNA and tRNA (C38 of tRNA Asp ). DNA methylation would play an important role in silencing transposable elements, providing stability to the amoeba genome [163][164][165][166]. ...
Protozoan parasite diseases cause significant mortality and morbidity worldwide. Factors such as climate change, extreme poverty, migration, and a lack of life opportunities lead to the propagation of diseases classified as tropical or non-endemic. Although there are several drugs to combat parasitic diseases, strains resistant to routinely used drugs have been reported. In addition, many first-line drugs have adverse effects ranging from mild to severe, including potential carcinogenic effects. Therefore, new lead compounds are needed to combat these parasites. Although little has been studied regarding the epigenetic mechanisms in lower eukaryotes, it is believed that epigenetics plays an essential role in vital aspects of the organism, from controlling the life cycle to the expression of genes involved in pathogenicity. Therefore, using epigenetic targets to combat these parasites is foreseen as an area with great potential for development. This review summarizes the main known epigenetic mechanisms and their potential as therapeutics for a group of medically important protozoal parasites. Different epigenetic mechanisms are discussed, highlighting those that can be used for drug repositioning, such as histone post-translational modifications (HPTMs). Exclusive parasite targets are also emphasized, including the base J and DNA 6 mA. These two categories have the greatest potential for developing drugs to treat or eradicate these diseases.
... Notably, conformation changes of important motifs, such as motif VI (ENV) and motif IV (PCQ) in DNMT2/TRDMT1 structure, can affect the enzymatic activity [19], while some elements in the tRNA cloverleaf structure determine the accuracy of tRNA discrimination by DNMT2/TRDMT1. E. histolytica DNA methyltransferase (Ehmeth), as a member of DNMT2/TRDMT1 family, can bind to the anticodon stem-loop (ACSL) construct of tRNA Asp-GUC , but tRNA full-length sequence is indispensable to the formation of a stable tRNA-Ehmeth complex in vitro [35]. In addition, an earlier study has demonstrated that the tRNA Asp-GUC ACSL construct alone does not serve as DNMT2/TRDMT1 substrate in vitro [12]. ...
Methylation is a common post-transcriptional modification of tRNAs, particularly in the anticodon loop region. The cytosine 38 (C38) in tRNAs, such as tRNAAsp-GUC, tRNAGly-GCC, tRNAVal-AAC, and tRNAGlu-CUC, can be methylated by human DNMT2/TRDMT1 and some homologs found in bacteria, plants, and animals. However, the substrate properties and recognition mechanism of DNMT2/TRDMT1 remain to be explored. Here, taking into consideration common features of the four known substrate tRNAs, we investigated methylation activities of DNMT2/TRDMT1 on the tRNAGly-GCC truncation and point mutants, and conformational changes of mutants. The results demonstrated that human DNMT2/TRDMT1 preferred substrate tRNAGly-GCC in vitro. L-shaped conformation of classical tRNA could be favourable for DNMT2/TRDMT1 activity. The complete sequence and structure of tRNA were dispensable for DNMT2/TRDMT1 activity, whereas T-arm was indispensable to this activity. G19, U20, and A21 in D-loop were identified as the important bases for DNMT2/TRDMT1 activity, while G53, C56, A58, and C61 in T-loop were found as the critical bases. The conserved CUXXCAC sequence in the anticodon loop was confirmed to be the most critical determinant, and it could stabilize C38-flipping to promote C38 methylation. Based on these tRNA properties, new substrates, tRNAVal-CAC and tRNAGln-CUG, were discovered in vitro. Moreover, a single nucleotide substitute, U32C, could convert non-substrate tRNAAla-AGC into a substrate for DNMT2/TRDMT1. Altogether, our findings imply that DNMT2/TRDMT1 relies on a delicate network involving both the primary sequence and tertiary structure of tRNA for substrate recognition.
... histolytica [23][24][25] incited us to check if queuine also enhances the level of m 5 C 38 in the parasite's tRNA Asp anticodon loop (Fig 1B). We used PCR-based bisulfite sequencing to detect cytosine methylation in the tRNA by identifying the location of the methylated cytosine by sequencing. ...
Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here we describe the effects of queuine on the physiology of the eukaryotic parasite, Entamoeba histolytica , the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C 38 in tRNA Asp GUC . Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in OS response, such as heat shock protein 70 (Hsp 70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C 38 in tRNA Asp GUC , parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.
Importance
Entamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of the bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces a mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.
... This experiment suggests either that Pmt1 directly interacts with Q 34 , or that Q 34 appropriately affects the ACL structure of tRNA Asp . The crystal structures of several Dnmt2 homologs have been solved, but unfortunately without the tRNA substrate (Dong et al. 2001;Schulz et al. 2012;Li et al. 2013). Based on a modeled tRNA Asp -Dnmt2 structure, it is also possible that Q 34 could alter the geometry of the ACL, allowing for better interactions between Dnmt2 and tRNA. ...
The numerous post-transcriptional modifications of tRNA play a crucial role in tRNA function. While most modifications are introduced to tRNA independently, several sets of modifications are found to be interconnected such that the presence of one set of modifications drives the formation of another modification. The vast majority of these modification circuits are found in the anticodon loop region where the largest variety and highest density of modifications occur compared to the other parts of the tRNA and where there is relatively limited sequence and structural information. We speculate here that the modification circuits in the anticodon loop region arise to enhance enzyme modification specificity by direct or indirect use of the first modification in the circuit as an additional recognition element for the second modification. We also describe the five well studied modification circuits in the anticodon loop, and outline possible mechanisms by which they may act. The prevalence of these modification circuits in the anticodon loop and the phylogenetic conservation of some of them suggest that a number of other modification circuits will be found in this region in different organisms.
... including loop regions, and exhibits an overall simiIar fold with the Dnmt2 crystal structures from human (hsD-nmt2) 16 and the pathogenic amoebae E. histolytica (ehDnmt2) 17 (Fig. 3b). In the spDnmt2 structure we were able to build a loop of 20 amino acids starting with Cys80, which was proved to be important for catalysis in hsDnmt2 and therefore is referred to this loop as the "active site loop". ...
... This loop is presumably unstructured in solution as the electron density for the corresponding amino acids was only observed for one of the four Dnmt2 molecules in the asymmetric unit, and this loop adopted a defined conformation due to formation of crystal contacts with another Dnmt2 molecule in the crystal lattice. A corresponding loop is also resolved in ehDnmt2 structure where it exhibits a different conformation which appears to be confined by crystal contacts 17 . The crystal structure of hsDnmt2 does not exhibit any traceable loop comprising the corresponding amino acids, arguing flexibility of the active site loop in Dnmt2 of all three organisms. ...
... Diffraction images were indexed, integrated and scaled using the XDS-package 30 . The structure of spDnmt2 in complex with SAH was solved by molecular replacement by PHASER 31 , as implemented in the CCP4 suite 32 , using the structure of the Entamoebae histolytica Dnmt2 homolog EhMeth (PBD-ID: 3QV2) 17 . ...
Dnmt2 methylates cytosine at position 38 of tRNAAsp in a variety of eukaryotic organisms. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNAAsp and the Dnmt2 dependent C38 methylation was recently found in vivo for S. pombe and D. discoideum. We demonstrate a direct effect of the Q-modification on the methyltransferase catalytic efficiency in vitro, as Vmax/K0.5 of purified S. pombe Dnmt2 shows an increase for in vitro transcribed tRNAAsp containing Q34 to 6.27 ∗ 10-3 s-1 µM-1 compared to 1.51 ∗ 10-3 s-1 µM-1 for the unmodified substrate. Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. In order to get insight into the structural basis for the Q-dependency, the crystal structure of S. pombe Dnmt2 was determined at 1.7 Å resolution. It closely resembles the known structures of human and E. histolytica Dnmt2, and contains the entire active site loop. The interaction with tRNA was analyzed by means of mass-spectrometry using UV cross-linked Dnmt2-tRNA complex. These cross-link data and computational docking of Dnmt2 and tRNAAsp reveal Q34 positioned adjacent to the S-adenosylmethionine occupying the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer.
... Our objectives in present work were to analyse the structural and physicochemical characteristics evolution of the DNMT2 in Drosophilidae comparing to Spodoptera frugiperda [26], human [27], Entamoeba histolytica [28], Haemophilus haemolyticus [29], [30], Haemophilus influenzae [31] DNMT2 and Geobacter sulfurreducens DNMT2 [18]. For this, we perform molecular homology modelling and determination of the electrostatic potential molecular surface of the DNMT2 enzymes. ...
... The search in the PDB database returned seventeen crystallographic structures of DNMT2 (S1 Table). In the present study, we used S. frugiperda [26], human [27], E. histolytica [28], H. haemolyticus [29] and H. influenzae [31] crystallographic models. ...
The amino acid sequence of DNMT2 is very similar to the catalytic domains of bacterial and eukaryotic proteins. However, there is great variability in the region of recognition of the target sequence. While bacterial DNMT2 acts as a DNA methyltransferase, previous studies have indicated low DNA methylation activity in eukaryotic DNMT2, with preference by tRNA methylation. Drosophilids are known as DNMT2-only species and the DNA methylation phenomenon is a not elucidated case yet, as well as the ontogenetic and physiologic importance of DNMT2 for this species group. In addition, more recently study showed that methylation in the genome in Drosophila melanogaster is independent in relation to DNMT2. Despite these findings, Drosophilidae family has more than 4,200 species with great ecological diversity and historical evolution, thus we, therefore, aimed to examine the drosophilids DNMT2 in order to verify its conservation at the physicochemical and structural levels in a functional context. We examined the twenty-six DNMT2 models generated by molecular modelling and five crystallographic structures deposited in the Protein Data Bank (PDB) using different approaches. Our results showed that despite sequence and structural similarity between species close related, we found outstanding differences when they are analyzed in the context of surface distribution of electrostatic properties. The differences found in the electrostatic potentials may be linked with different affinities and processivity of DNMT2 for its different substrates (DNA, RNA or tRNA) and even for interactions with other proteins involved in the epigenetic mechanisms.
... Genome methylation in D. melanogaster does not require DNMT2 suggesting the presence of a novel DNMT (Takayama et al. 2014). In E. histolytica, a DNMT2-like methyltransferase, which is responsible for DNA cytosine methylation, appears to methylate both specific DNA and tRNA targets (Fisher et al. 2006;Schulz et al. 2012). ...
To date, little is known about cytosine methylation in the genomic DNA of apicomplexan parasites, although it has been confirmed that this important epigenetic modification exists in many lower eukaryotes, plants, and animals. In the present study, ELISA-based detection demonstrated that low levels of 5-methylcytosine (5-mC) are present in Eimeria spp., Toxoplasma gondii, Cryptosporidium spp., and Neospora caninum. The proportions of 5-mC in genomic DNA were 0.18???0.02% in E tenella sporulated oocysts, 0.19???0.01% in E. tenella second-generation merozoites, 0.22???0.04% in T. gondii tachyzoites, 0.28???0.03% in N. caninum tachyzoites, and 0.06???0.01, 0.11???0.01, and 0.09???0.01% in C. andersoni, C. baileyi, and C. parvum sporulated oocysts, respectively. In addition, we found that the percentages of 5-mC in E. tenella varied considerably at different life stages, with sporozoites having the highest percentage of 5-mC (0.78???0.10%). Similar stage differences in 5-mC were also found in E. maxima, E. necatrix, and E. acervulina, the levels of 5-mC in their sporozoites being 4.3-, 1.8-, 2.5-, and 2.0-fold higher than that of sporulated oocysts, respectively (p?<?0.01). Furthermore, a total DNA methyltransferase-like activity was detected in whole cell extracts prepared from E. tenella sporozoites. In conclusion, genomic DNA methylation is present in these apicomplexan parasites and may play a role in the stage conversion of Eimeria.
... It will be interesting to determine how the presence of Q in tRNA stimulates Dnmt2 activity. The crystal structure of several Dnmt2 homologs has been determined, but unfortunately only without the tRNA substrate [35][36][37]. However, in one study, information on the effect of point mutations in Dnmt2 on tRNA binding and enzymatic activity was used to model tRNA Asp binding on the Dnmt2 structure [38]. ...
Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA) methyltransferases for cytosine-5 methylation, foremost C38 (m5C38) of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe. Significantly, the presence of the nucleotide queuosine in tRNAAsp strongly stimulates Dnmt2 activity both in vivo and in vitro in S. pombe. Queuine, the respective base, is a hypermodified guanine analog that is synthesized from guanosine-5’-triphosphate (GTP) by bacteria. Interestingly, most eukaryotes have queuosine in their tRNA. However, they cannot synthesize it themselves, but rather salvage it from food or from gut microbes. The queuine obtained from these sources comes from the breakdown of tRNAs, where the queuine ultimately was synthesized by bacteria. Queuine thus has been termed a micronutrient. This review summarizes the current knowledge of Dnmt2 methylation and queuosine modification with respect to translation as well as the organismal consequences of the absence of these modifications. Models for the functional cooperation between these modifications and its wider implications are discussed.
... To date, crystal structures of three Dnmt2 enzymes have been deposited in the protein database (PDB), namely from human (entry 1G55) 12 , Spodoptera frugiperda 15 (4H0N) and from Entamoeba histolytica (3QV2) 16 . The latter structure is shown in Figure 2, next to a structure of tRNA Asp GUC (1VTQ). ...
... These missing features are more clearly discernible in the S. frugiperda structure, but this enzyme contains a significantly shorter loop. The active site loop (residues 80--100) is better defined in the third structure of Ehmeth, the Dnmt2 homolog of E. histolytica16 , which was solved by the Ficner and Ankri groups. Specifically, the crystal structure of Ehmeth revealed that this loop adopts an α-helical conformation. ...
A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.
