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Many paths to methyltransfer: A chronicle of convergence
Abstract
S-adenosyl-L-methionine (AdoMet) dependent methyltransferases (MTases) are involved in biosynthesis, signal transduction, protein repair, chromatin regulation and gene silencing. Five different structural folds (I-V) have been described that bind AdoMet and catalyze methyltransfer to diverse substrates, although the great majority of known MTases have the Class I fold. Even within a particular MTase class the amino-acid sequence similarity can be as low as 10%. Thus, the structural and catalytic requirements for methyltransfer from AdoMet appear to be remarkably flexible.
... Methyltransferases with the Rossmann-like fold are also known as Class I methyltransferases and possess seven β-strands with the topology ↑3-↑2-↑1-↑4-↑5-↓7-↑6 (Schubert et al. 2003). β-strands 1-3 of Class I RNA methyltransferases serve as a platform for structural elements that bind the Sadenosylmethionine cofactor while β-strands 4-7 play the same role for RNA binding (Fig. 1A.) ...
... /2024 In all cases RRAD proteins are thought to bind S-adenosylmethionine (SAM) and the nucleotide targeted for methylation in a similar manner using conserved motifs (Fig. 1D). RRAD proteins contain conserved motifs observed in other 6-methyladenosine methyltransferases (Schubert et al. 2003). These include Motif I, a Gly-rich region that sits adjacent to the kink between the ribose and methionyl moieties of SAM and has shape complementarity to it. ...
The Ribosomal RNA Adenine Dimethylase (RRAD) family of enzymes facilitate ribosome maturation in all organisms by dimethylating two nucleotides of small subunit rRNA. Prominent members of this family are the human DIMT1 and bacterial KsgA enzymes. A sub-group of RRAD enzymes, named erythromycin resistance methyltransferases (Erm) dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays we found that the N-terminal region is critical for activity in RRAD enzymes: the N-terminal basic region promotes rRNA binding and the conserved motif likely assists in juxtaposing the adenosine substrate and the SAM cofactor. Our results and emerging structural data suggest this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding forming a cap on the active site required for methylation.
... S-adenosyl-L-methionine (SAM)-dependent methyltransferases are classified according to the structure of catalytic domain (29). Although the majority of tRNA methyltransferases are classified as Class I enzymes, TrmH belongs to the Class IV enzymes (29). ...
... S-adenosyl-L-methionine (SAM)-dependent methyltransferases are classified according to the structure of catalytic domain (29). Although the majority of tRNA methyltransferases are classified as Class I enzymes, TrmH belongs to the Class IV enzymes (29). The Class IV enzymes are known as SpoU-TrmD (SPOUT) superfamily enzymes (30)(31)(32) with SpoU being the classical name of TrmH (21,22). ...
TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2’-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA SerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyrand tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.
... Once the methyl group is donated, the resulting S -adenosyl-L -homocysteine (SAH) is degraded due to its toxicity ( 15 , 16 ). To maintain SAM concentration, SAM acts as an inhibitor of MetK synthesis (17)(18)(19)(20) . This regulation is achie v ed by the SAM-riboswitches, which bind SAM and acts as negati v e feedback unit for genes in methionine or cysteine biosynthesis. ...
... Six sub-classes of SAM riboswitches (SAM-I to SAM-VI) have been identified, classified into three families according to their structural features (17)(18)(19)(20)(21)(22)(23)(24)(25). In general, SAM riboswitches exhibit strong discrimination between SAM and SAH by electrostatically interacting with the positi v e-charged sulfonium cation of the SAM molecule (26)(27)(28)(29)(30)(31)(32)(33), as previously shown by X-ray crystallography and single-molecule methods ( 22 , 34 ). ...
Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.
... Two negatively charged aspartates, Asp88 and Asp301, form hydrogen bonds respectively with the adenosyl N6 amino group and the two hydroxyl oxygen atoms of the sinefungin ribose (Fig. 8D). The Asp301 interactions are consistent with expectations for a Class I MTase (109). In addition, His253 forms an H-bond with one of the ribose hydroxyl oxygen atoms. ...
... Substituting the adenine with a cytosine in silico resulted in a model implying that the cytosine ring stacks with Tyr110 of the N4CMT SPPY motif, while the polar edge of the cytosine (O2, N3, and N4) occupies (or is near to) the water positions #3 (O2), #2 (N3), and #1 (N4) (Fig. 8J). This arrangement positions the target N4 atom in line with the transferrable methyl group and sulfur atom of SAM (in the place of sinefungin), consistent with the S N 2 reaction mechanism used by SAMdependent MTases (109,110). ...
Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (a/c)CG(t/c/a), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemi-methylated intermediates and eventually fully-methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle-regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle-regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis-dependent methylation following DNA replication.
... The activity of methyltransferases has been demonstrated against a broad range of substrates including Frontiers in Oncology frontiersin.org 01 OPEN ACCESS EDITED BY nucleic acids (DNA/RNA), proteins, lipids, and small molecules (4)(5)(6). Arguably the most researched form of methylation is performed by DNA methyltransferases. Both the enzymes that methylate the DNA and the proteins that recognize this modification play critical roles in normal development through their ability to modulate transcriptional activity (e.g., gene silencing through the hypermethylation of gene promoter regions) (5). ...
... The best-characterized members of the METTL family are METTL3 and METTL14, which form a heterodimer capable of catalyzing the N 6 -methyladenosine modification (m 6 A) on adenosine residues in RNA molecules (12). This modification can in turn be recognized by a wide range of modification "readers" that can influence transcript splicing, transport, stability, and translation (6). Other members of the METTL family have subsequently been shown to catalyze not only other nucleotide modifications such as N 4 -methylcytidine (METTL15) or O 2 -methyluracil (METTL19) but to also methylate protein substrates on different residues (METTL10, METTL11A/B, METTL20) (8,11). ...
Methyltransferases are enzymes fundamental to a wide range of normal biological activities that can become dysregulated during oncogenesis. For instance, the recent description of the methyltransferase-like (METTL) family of enzymes, has demonstrated the importance of the N⁶-adenosine-methyltransferase (m⁶A) modification in transcripts in the context of malignant transformation. Because of their importance, numerous METTL family members have been biochemically characterized to identify their cellular substrates, however some members such as METTL7B, recently renamed TMT1B and which is the subject of this review, remain enigmatic. First identified in the stacked Golgi, TMT1B is also localized to the endoplasmic reticulum as well as lipid droplets and has been reported as being upregulated in a wide range of cancer types including lung cancer, gliomas, and leukemia. Interestingly, despite evidence that TMT1B might act on protein substrates, it has also been shown to act on small molecule alkyl thiol substrates such as hydrogen sulfide, and its loss has been found to affect cellular proliferation and migration. Here we review the current evidence for TMT1B’s activity, localization, and potential biological role in the context of both normal and cancerous cell types.
... The C terminus of ASPL is sufficient to provide monomeric p97 for VCPKMT INTRODUCTION Methyltransferases (MTases) catalyze the transfer of a methyl group from an S-adenosyl-L-methionine (SAM) to various substrates, such as proteins, DNA, RNA, lipids, and small molecules, which are involved in biosynthesis, cell signaling, protein repair, and gene regulation. [1][2][3] Among the five different MTase classes (I-V), the majority of known MTases belong to class I MTases, which are also designated as the SET (Su(var)3-9), enhancer-of-zeste, trithorax) domain proteins. 3 Most lysine-specific class I MTases catalyze the transfer to histones, and histone-lysine methyltransferase (DOT1L) has been studied the most. ...
... [1][2][3] Among the five different MTase classes (I-V), the majority of known MTases belong to class I MTases, which are also designated as the SET (Su(var)3-9), enhancer-of-zeste, trithorax) domain proteins. 3 Most lysine-specific class I MTases catalyze the transfer to histones, and histone-lysine methyltransferase (DOT1L) has been studied the most. 4-10 A few class I MTases methylate lysine or arginine on non-histones and mainly recognize specific motifs in unstructured polypeptides. ...
p97 is a human AAA+ (ATPase associated with diverse cellular activities, also known as valosin-containing protein [VCP]) ATPase, which is involved in diverse cellular processes such as membrane fusion and proteolysis. Lysine-specific methyltransferase of p97 (METTL21D) was identified as a class I methyltransferase that catalyzes the trimethylation of Lys315 of p97, a so-called VCP lysine methyltransferase (VCPKMT). Interestingly, VCPKMT disassembles a single hexamer ring consisting of p97-D1 domain and methylates Lys315 residue. Herein, the structures of S-adenosyl-L-methionine-bound VCPKMT and S-adenosyl-L-homocysteine-bound VCPKMT in complex with p97 N/D1 (N21-Q458) were reported at a resolution of 1.8 Å and 2.8 Å, respectively. The structures revealed the molecular details for the recognition and methylation of monomeric p97 by VCPKMT. Using biochemical analysis, we also investigated whether the methylation of full-length p97 could be sufficiently enhanced through cooperation between VCPKMT and the C terminus of alveolar soft part sarcoma locus (ASPL). Our study provides the groundwork for future structural and mechanistic studies of p97 and inhibitors.
... Specifically, the pathway only contained one gene, SETIT_015292mg, which encodes a methyltransferase involved in the post-modification of flavonoids. The gene clearly encodes flavonoid O-methyltransferase (FOMT), which serves as a key modifying enzyme in the flavonoid metabolic pathway by catalyzing the synthesis of O-methylated derivatives of flavonoids [63]. Furthermore, SETIT_015292mg also plays a crucial role in the stilbenoid, diarylheptanoid, and gingerol biosynthesis pathway (Table S8). ...
The grain filling rate (GFR) plays a crucial role in determining grain yield. However, the regulatory and molecular mechanisms of the grain filling rate (GFR) in foxtail millet remains unclear. In this study, we found that the GFR of ′Changnong No.47′ (CN47) was significantly higher at 14 DAF (days after flowering) and 21 DAF in comparison to ′Changsheng 13′ (CS13). Furthermore, CN47 also exhibited higher a thousand-grain weight and yield than CS13. Therefore, RNA-seq and UHPLC-MS/MS were used to conduct transcriptome and metabolome analyses during two stages of grain filling in both cultivars. Conjoint analysis of transcriptomics and metabolomics was adopted in order to analyze the biological processes and functional genes associated with GFR. The results identified a total of 765 differentially expressed genes (DEGs) and 246 differentially accumulated metabolites (DAMs) at the 14 DAF stage, while at the 21 DAF stage, a total of 908 DEGs and 268 DAMs were identified. The integrated analysis of co-mapped DAMs and DEGs revealed enriched pathways, including flavonoid biosynthesis, plant hormone signal transduction, tyrosine metabolism, ATP-binding cassette (ABC) transporters, and beta-Alanine metabolism, as well as stilbenoid, diarylheptanoid, and gingerol biosynthesis. In order to elucidate their potential functions in the context of GFR, we developed a gene–metabolite regulatory network for these metabolic pathways. Notably, we found that some genes associated with ABC transporters and the plant hormone signal transduction pathway were implicated in auxin transport and signal transduction, highlighting the crucial role of auxin during grain filling. These findings provide initial insights into the regulatory and molecular mechanisms underlying GFR in foxtail millet, as well as offering valuable genetic resources for further elucidation of GFR in future studies. The findings have also established a theoretical basis for improving the efficiency of yield breeding in foxtail millet.
... A. fumigatus Erg6 and Smt1 proteins are predicted to harbor a conserved methyltransferase domain orchestrating SAM binding, with an identity of 71% and 37%, respectively, when aligned with methyltransferase domain from S. cerevisiae (Fig. S1B). Additionally, Erg6 of A. fumigatus and S. cerevisiae are both predicted to encode a conserved sterol methyltransferase C-terminal domain, which is responsible for selective substrate binding 23,24 . This conserved substrate-binding region was not found in A. fumigatus Smt1 (Fig. S1B). ...
Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target.
... Specifically, the pathway only contained one gene, SETIT_015292mg, which encoded a methyltransferase involved in the post-modification of flavonoids. The gene clearly encoded flavonoid O-methyltransferase (FOMT), which serves as a key modifying enzyme in the flavonoid metabolic pathway by catalyzing the synthesis of O-methylated derivatives of flavonoids [63]. Furthermore, SETIT_015292mg also played a crucial role in the stilbenoid, diarylheptanoid and gingerol biosynthesis pathway (Table S8). ...
The grain filling rate (GFR) plays a crucial role in determining grain yield. However, the regulatory and molecular mechanism of grain filling rate (GFR) in foxtail millet remains unclear. In this study, we found the GFR of 'Changnong No.47' (CN47) was significantly higher at 14 days after flowering (DAF) and 21 DAF in comparison to 'Changsheng 13' (CS13). Furthermore, CN47 also exhibited higher thousand grain weight and yield than CS13. Therefore, we conducted transcriptomics and metabolomics to analyze the biological processes and functional genes associated with GFR during two stages of grain filling in both cultivars. A total of 765 differentially expressed genes (DEGs) and 246 differentially accumulated metabolites (DAMs) were identified at the 14 DAF stage, while at the 21 DAF stage, a total of 908 DEGs and 268 DAMs were identified. The integrated analysis of co-mapped DAMs and DEGs revealed enriched pathways, including flavonoid biosynthesis, plant hormone signal transduction, tyrosine metabolism, ATP-binding cassette (ABC) transporters, beta-alanine metabolism, as well as stilbenoid, diarylheptanoid and gingerol biosynthesis. In order to elucidate their potential functions in the context of GFR, we developed a gene-metabolite regulatory network for these metabolic pathways. Notably we found some genes associated with ABC transporters and plant hormone signal transduction pathway were implicated in auxin transport and signal transduction, highlighting the crucial role of auxin during grain filling. The results provide initial insights into the regulatory and molecular mechanisms underlying GFR in foxtail millet, as well as offer valuable genetic resources for further elucidation of GFR in future studies. The findings have established a theoretical basis for improving the efficiency of yield breeding in foxtail millet.
... Northern-blot assays were used to determine the orientation of the motif, proving it resides upstream of the smtA gene (Breaker Laboratory, unpublished findings). The smtA gene encodes a class I SAM-dependent methyltransferase (MTase), which uses S -adenosyl-L-methionine (SAM) as methyl donor ( 95 ). Class I SAM MTases represent the largest of the five classes of these enzymes ( 96 ), making it difficult to associate the motif with a specific metabolic pathway or biological process. ...
Structured noncoding RNAs (ncRNAs) contribute to many important cellular processes involving chemical catalysis, molecular recognition and gene regulation. Few ncRNA classes are broadly distributed among organisms from all three domains of life, but the list of rarer classes that exhibit surprisingly diverse functions is growing. We previously developed a computational pipeline that enables the near-comprehensive identification of structured ncRNAs expressed from individual bacterial genomes. The regions between protein coding genes are first sorted based on length and the fraction of guanosine and cytidine nucleotides. Long, GC-rich intergenic regions are then examined for sequence and structural similarity to other bacterial genomes. Herein, we describe the implementation of this pipeline on 50 bacterial genomes from varied phyla. More than 4700 candidate intergenic regions with the desired characteristics were identified, which yielded 44 novel riboswitch candidates and numerous other putative ncRNA motifs. Although experimental validation studies have yet to be conducted, this rate of riboswitch candidate discovery is consistent with predictions that many hundreds of novel riboswitch classes remain to be discovered among the bacterial species whose genomes have already been sequenced. Thus, many thousands of additional novel ncRNA classes likely remain to be discovered in the bacterial domain of life.
... This indicates that all of them use SAM as a cofactor in their enzymatic reactions. Delineation of these classes is based on structural attributes of their catalytic domains: SAM-OMTs of class I feature a Rossmann-like alpha/beta structure, while SAM-OMTs of class II show a TIM beta/alpha-barrel structure [55]. Moreover, the plant OMTs depicted in 5 were identified as caffeic acid OMT-like, indicating a potential substrate affinity for caffeic acid. ...
Background
Floral scents play a crucial role in attracting insect pollinators. Among the compounds attractive to pollinators is 1,4-dimethoxybenzene (1,4-DMB). It is a significant contributor to the scent profile of plants from various genera, including economically important Cucurbita species. Despite its importance, the biosynthetic pathway for the formation of 1,4-DMB was not elucidated so far.
Results
In this study we showed the catalysis of 1,4-DMB in the presence of 4-methoxyphenol (4-MP) by protein extract from Styrian oil pumpkin (Cucurbita pepo) flowers. Based on this finding, we identified a novel O-methyltransferase gene, Cp4MP-OMT, whose expression is highly upregulated in the volatile-producing tissue of pumpkin flowers when compared to vegetative tissues. OMT activity was verified by purified recombinant Cp4MP-OMT, illustrating its ability to catalyse the methylation of 4-MP to 1,4-DMB in the presence of cofactor SAM (S-(5′-adenosyl)-L-methionine).
Conclusions
Cp4MP-OMT is a novel O-methyltransferase from C. pepo, responsible for the final step in the biosynthesis of the floral scent compound 1,4-DMB. Considering the significance of 1,4-DMB in attracting insects for pollination and in the further course fruit formation, enhanced understanding of its biosynthetic pathways holds great promise for both ecological insights and advancements in plant breeding initiatives.
... Biochemical-metabolism-related MTases have complex functions with diverse methyl acceptors, ranging from inorganic molecules to biological macromolecules, such as proteins, hormones, amine compounds, flavonoids, chlorophyll, tocopherols, etc. (Fernandez-Pozo et al. 2015). Based on distinct binding sites, MTases can be also classified into O-MTases, C-MTases, N-MTases, S-MTases, and inorganic arsenic MTases (Cyt19) (Schubert et al. 2003;Dong et al. 2018). ...
Main conclusion
Six methyltransferase genes affecting tomato fruit ripening were identified through genome-wide screening, VIGS assay, and expression pattern analysis. The data provide the basis for understanding new mechanisms of methyltransferases.
Abstract
Fruit ripening is a critical stage for the formation of edible quality and seed maturation, which is finely modulated by kinds of factors, including genetic regulators, hormones, external signals, etc. Methyltransferases (MTases), important genetic regulators, play vital roles in plant development through epigenetic regulation, post-translational modification, or other mechanisms. However, the regulatory functions of numerous MTases except DNA methylation in fruit ripening remain limited so far. Here, six MTases, which act on different types of substrates, were identified to affect tomato fruit ripening. First, 35 MTase genes with relatively high expression at breaker (Br) stage of tomato fruit were screened from the tomato MTase gene database encompassing 421 genes totally. Thereafter, six MTase genes were identified as potential regulators of fruit ripening via virus-induced gene silencing (VIGS), including four genes with a positive regulatory role and two genes with a negative regulatory role, respectively. The expression of these six MTase genes exhibited diverse patterns during the fruit ripening process, and responded to various external ripening-related factors, including ethylene, 1-methylcyclopropene (1-MCP), temperature, and light exposure. These results help to further elaborate the biological mechanisms of MTase genes in tomato fruit ripening and enrich the understanding of the regulatory mechanisms of fruit ripening involving MTases, despite of DNA MTases.
... The origin of resistance genes from functional changes is illustrated by the very ancient evolution of beta-lactamases from penicillin-binding proteins or the appearance of erm-family 23S rRNA methylases, which have been shown to be an important mechanism of macrolide/lincosamide/Streptogramin B resistance [25,26]. The ancestors of these methylases are housekeeping genes associated with ribosome biogenesis and the regulation of ribosomal functions [27,28]. It has been experimentally demonstrated that these genes are capable of undergoing changes that lead to macrolide resistance [29]. ...
Oxazolidinone resistance, especially transmissible resistance, is a major public health concern, and the origin of this resistance mechanism is not yet resolved. This study aims to delve into the phylogenetic origin of the transmissible oxazolidinone resistance mechanisms conferring cross-resistance to other drugs of human and veterinary importance. The amino acid sequences of the five cfr ribosomal methylases and optrA and poxtA were used as queries in searches against 219,549 bacterial proteomes in the NCBI RefSeq database. Hits with > 40% amino acid identity and > 80% query coverage were aligned, and phylogenetic trees were reconstructed. All five cfr genes yielded highly similar trees, with rlmN housekeeping ribosomal methylases located basal to the sister groups of S-adenosyl-methionine-dependent methyltransferases from various Deltaproteobacteria and Actinomycetia, including antibiotic-producing Streptomyces species, and the monophyletic group of cfr genes. The basal branches of the latter contained paenibacilli and other soil bacteria; they then could be split into the clades [cfr(C):cfr(E)] and [[cfr:cfr(B)]:cfr(D)], always with different Bacillaceae in their stems. Lachnospiraceae were encountered in the basal branches of both optrA and poxtA trees. The ultimate origin of the cfr genes is the rlmN housekeeping ribosomal methylases, which evolved into a suicide-avoiding methylase in antibiotic producers; a soil organism (Lachnospiraceae, Paenibacilli) probably acted as a transfer organism into pathogenic bacteria. In the case of optrA, the porcine pathogenic Streptococcus suis was present in all branches, while the proteins closest to poxtA originated from Clostridia.
... Consistent with predictions 18,19 , the structure of PsiM (Fig. 2a, b) comprises a Rossmann fold characteristic of class I SAM-dependent methyltransferases 23 . This fold consists of 6 parallel β-strands, interlaced with α-helices and followed by a single antiparallel strand. ...
Psilocybin, the natural hallucinogen produced by Psilocybe (“magic”) mushrooms, holds great promise for the treatment of depression and several other mental health conditions. The final step in the psilocybin biosynthetic pathway, dimethylation of the tryptophan-derived intermediate norbaeocystin, is catalysed by PsiM. Here we present atomic resolution (0.9 Å) crystal structures of PsiM trapped at various stages of its reaction cycle, providing detailed insight into the SAM-dependent methylation mechanism. Structural and phylogenetic analyses suggest that PsiM derives from epitranscriptomic N⁶-methyladenosine writers of the METTL16 family, which is further supported by the observation that bound substrates physicochemically mimic RNA. Inherent limitations of the ancestral monomethyltransferase scaffold hamper the efficiency of psilocybin assembly and leave PsiM incapable of catalysing trimethylation to aeruginascin. The results of our study will support bioengineering efforts aiming to create novel variants of psilocybin with improved therapeutic properties.
... The SAMMTase family catalyses the transfer of methyl groups from SAM to a plethora of molecules, including DNA, RNA, proteins and so on, thereby modulating various activities, including biosynthesis, signal transduction, protein repair, chromatin regulation and gene silencing (Schubert et al., 2003;Struck et al., 2012). DNA methylation is one of the insect epigenetics that has received extensive attention (Jaenisch and Bird, 2003;Field et al., 2004;Lyko and Maleszka, 2011;Mandrioli and Manicardi, 2015). ...
S-Adenosyl-l-methionine-dependent methyltrans-ferases (SAMMTases) modulate important cellular and metabolic activities in both prokaryotes and eukaryotes. Here, we functionally characterized an SAMMTase gene (MTase15) in the migratory brown planthopper (BPH), Nilaparvata lugens, which is the most notorious rice pest in Asia. The cDNA sequence of MTase15 is 2764 nt in length with an open reading frame of 1218 nt encoding 405 amino acid residues. Quantitative real-time PCR analysis showed that MTase15 was readily detected from egg to adult stages and extensively distributed in various body parts of adult females and males, with slightly high levels in ovary and testis, respectively. In addition, MTase15 was transcriptionally regulated by the insulin signalling pathway in BPH. RNA-interference-mediated knockdown of MTase15 (dsMtase15) resulted in deficiencies in vitellogenin synthesis and oogenesis, and female infertility. Males with Mtase15 knockdown retained the capability of producing sperms with normal viability, but less sperm was transferred to wild-type (wt) females during copulation, and eggs laid by these wt females arrested embryogenesis. These findings not only assign a functional role to MTase15, but also provide a link between the insulin signalling pathway and epigenetic regulation in BPH reproduction.
... Methyltransferase-like (METTL) family proteins are characterized by a conserved Rossman-like fold S-adenosyl methionine (SAM)-binding domain, which methylates proteins, nucleic acids, and other small molecule metabolites, are involved in the regulation of mRNA stability and translation efficiency [18][19][20] . Several METTLs localize to mitochondria, among which METTL9, METTL12 and METTL20 are responsible for protein methylation, while METTL8 and METTL2A for mt-tRNA methylation, METTL15 for mt-rRNA methylation 10,[21][22][23] . ...
Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The m ⁴ C methyltransferase METTL15 maintains mitochondrial homeostasis by catalyzing m ⁴ C839 located in 12 S rRNA helix 44 (h44). This modification is essential to fine-tuning the ribosomal decoding center and increasing decoding fidelity according to studies of a conserved site in Escherichia coli . Here, we reported a series of crystal structures of human METTL15–hsRBFA–h44–SAM analog, METTL15–hsRBFA–SAM, METTL15–SAM and apo METTL15. The structures presented specific interactions of METTL15 with different substrates and revealed that hsRBFA recruits METTL15 to mitochondrial small subunit for further modification instead of 12 S rRNA. Finally, we found that METTL15 deficiency caused increased reactive oxygen species, decreased membrane potential and altered cellular metabolic state. Knocking down METTL15 caused an elevated lactate secretion and increased levels of histone H4K12-lactylation and H3K9-lactylation. METTL15 might be a suitable model to study the regulation between mitochondrial metabolism and histone lactylation.
... 17,18 Biologically, methylation is performed by a family of enzymes known as lysine methyltransferases. 19 However, methylation can also be performed reductively using formaldehyde under mild conditions that do not alter the protein's structure. 20 This alteration causes a distinct change in the mass of the protein, providing a unique signature that can be detected through mass spectrometry (MS). ...
... However, designing small molecule inhibitors that target specific protein methyltransferases has remained challenging due to selectivity issues resulting in off-target effects in this large class of enzymes, and few compounds have made it to human trials and been approved by the FDA [28,[37][38][39][40][41][42]. SAM is a conformationally flexible molecule and can adopt numerous conformations, as observed in crystal structures of different classes of SAM-dependent methyltransferases [43]. Additionally, NMR data indicates that SAM is multi-conformeric when free in solution and the molecule can adopt multiple energetically reasonable physical conformations when bound with methyltransferase active sites, underscoring its conformational dynamics [44][45][46][47][48][49]. ...
... The enzymatically active domain of all KMT2 proteins is the highly conserved SET domain (Figure 2) (Schubert et al., 2003) located at the 3′ end of the gene (amino acids 4771-4893 of 4911 encoded by NM_170606.2). All reported PTC-inducing mutations that result in Kleefstra syndrome 2, including the case detailed in this report, (if translated) would truncate the SET domain, eradicating the methyltransferase activity of the truncated protein. ...
Background
Haploinsufficiency of the Lysine Methyltransferase 2C (KMT2C) gene results in the autosomal dominant disorder, Kleefstra syndrome 2. It is an extremely rare neurodevelopmental condition, with 14 previous reports describing varied clinical manifestations including dysmorphic features, delayed psychomotor development and delayed growth.
Methods
Here, we describe a female with global developmental delay, attention deficit disorder, dyspraxia, short stature and subtle non‐specific dysmorphic features. To identify causative mutations, whole exome sequencing was performed on the proband and her younger brother with discrete clinical presentation.
Results
Whole exome sequencing identified a novel de novo heterozygous 11 bp deletion in KMT2C (c.1759_1769del), resulting in a frameshift mutation and early termination of the protein (p.Gln587SerfsTer7). This variant is the second‐most N‐terminal reported mutation, located 4171 amino acids upstream of the critical enzymatically active SET domain (required for chromatin modification and histone methylation).
Conclusion
The majority of the other reported mutations are frameshift mutations upstream of the SET domain and are predicted to result in protein truncation. It is thought that truncation of the SET domain, results functionally in an inability to modify chromatin through histone methylation. This report expands the clinical and genetic characterisation of Kleefstra syndrome 2.
... Protein methyltransferases are classified into five classes, I, II, III, IV, and V, according to structural similarities of the catalytic domain 22 . PKMTs predominantly belong to the class V superfamily, containing a conserved Su(var)3-9, enhancer of zeste, and trithorax (SET) domain. ...
Protein lysine methyltransferases (PKMTs) play crucial roles in histone and nonhistone modifications, and their dysregulation has been linked to the development and progression of cancer. While the majority of studies have focused on the oncogenic functions of PKMTs, extensive evidence has indicated that these enzymes also play roles in tumor suppression by regulating the stability of p53 and β-catenin, promoting α-tubulin-mediated genomic stability, and regulating the transcription of oncogenes and tumor suppressors. Despite their contradictory roles in tumorigenesis, many PKMTs have been identified as potential therapeutic targets for cancer treatment. However, PKMT inhibitors may have unintended negative effects depending on the specific cancer type and target enzyme. Therefore, this review aims to comprehensively summarize the tumor-suppressive effects of PKMTs and to provide new insights into the development of anticancer drugs targeting PKMTs.
... The methyltransferases form a major superfamily, but in addition there are several other proteins that recruit SAM ligands regularly for methylation of biomolecules. Methyltransferases (MTases) themselves have been grouped into 5 distinct structural classes based on their catalytic domains, which are structurally different from one another (Rossmann-like α/β, TIM barrel, tetrapyrrole methylase α/β, SPOUT α/β and SET domain β folds) (Schubert et al., 2003). Even within a given class, the sequence similarity between proteins are reported to be as low as 10%. ...
S-adenosylmethionine (SAM) is a ubiquitous co-factor that serves as a donor for methylation reactions and additionally serves as a donor of other functional groups such as amino and ribosyl moieties in a variety of other biochemical reactions. Such versatility in function is enabled by the ability of SAM to be recognized by a wide variety of protein molecules that vary in their sequences and structural folds. To understand what gives rise to specific SAM binding in diverse proteins, we set out to study if there are any structural patterns at their binding sites. A comprehensive analysis of structures of the binding sites of SAM by all-pair comparison and clustering, indicated the presence of 4 different site-types, only one among them being well studied. For each site-type we decipher the common minimum principle involved in SAM recognition by diverse proteins and derive structural motifs that are characteristic of SAM binding. The presence of the structural motifs with precise three-dimensional arrangement of amino acids in SAM sites that appear to have evolved independently, indicates that these are winning arrangements of residues to bring about SAM recognition. Further, we find high similarity between one of the SAM site types and a well known ATP binding site type. We demonstrate using in vitro experiments that a known SAM binding protein, HpyAII.M1, a type 2 methyltransferase can bind and hydrolyse ATP. We find common structural motifs that explain this, further supported through site-directed mutagenesis. Observation of similar motifs for binding two of the most ubiquitous ligands in multiple protein families with diverse sequences and structural folds presents compelling evidence at the molecular level in favour of convergent evolution.
... [16,17] Structurally, COMT is a monomeric enzyme featuring a central ß-sheet flanked by α-helices on both sides. [14,18] The proposed mechanism of COMT-catalyzed methylation of catechols in the presence of SAM includes the coordination of Mg 2 + by the hydroxyl groups of the catechol substrate which lowers their pK a values; this aids the deprotonation of the hydroxyl group closest to the SAM methyl group by a lysine residue ( Figure 2). [14,19] The broad substrate range of COMTs can be explained by the architecture of the acceptor substrate binding pocket; apart from the Mg 2 + stabilizing the catechol moiety, the binding site is an open cleft fitting small and large catechol compounds such as 3,4-dihydroxybenzaldehyde and tetrahydroisoquinolines (THIQs), respectively. ...
S‐Adenosyl‐l‐methionine (SAM)‐dependent methyltransferases (MTs) are highly chemoselective enzymes grouped in C‐, N‐, O‐, S‐ and halide MTs, depending on the (hetero) atom that acts as the methyl group acceptor. So far, OMTs present the largest group, including many well investigated candidates. The catechol OMT from mammals such as from Rattus norvegicus (RnCOMT) is involved in the metabolism of neurotransmitters like dopamine. It is known to methylate the hydroxyl of the catechol ring in the 3 position. There are also reports showing that the regioselectivity of different COMTs can vary leading to different products with methyl groups in the 3 and or 4 positions. Nevertheless, there was only O‐methylation reported for COMTs. Another related MT, the caffeate OMT involved in the lignin biosynthesis of plants has also been reported as a chemoselective enzyme. In nature, S‐methylation is a rare phenomenon with different methyl donors being involved in the methyl transfer onto sulfur atoms. Several SAM‐dependent MTs are identified as S‐methyltransferases (SMTs), these are involved in salvaging pathways and xenobiotic metabolism of cells. Here, we report a new function of three OMTs; RnCOMT, a COMT from Myxococcus xanthus (MxSafC), and a CaOMT from Prunus persica (PpCaOMT) with acceptance towards different aromatic thiol substrates with up to full conversion.
... This analysis showed that MoRRP8 also belongs to class I methyltransferase, of which all members function as rRNA methyltransferase. The class I to V of AdoMet-dependent families were distinguished by having five structurally different folds [20]. ...
The nucleolus is the largest, membrane-less organelle within the nucleus of eukaryotic cell that plays a critical role in rRNA transcription and assembly of ribosomes. Recently, the nucleolus has been shown to be implicated in an array of processes including the formation of signal recognition particles and response to cellular stress. Such diverse functions of nucleolus are mediated by nucleolar proteins. In this study, we characterized a gene coding a putative protein containing a nucleolar localization sequence (NoLS) in the rice blast fungus, Magnaporthe oryzae. Phylogenetic and domain analysis suggested that the protein is orthologous to Rrp8 in Saccharomyces cerevisiae. MoRRP8-GFP (translational fusion of MoRRP8 with green fluorescence protein) co-localizes with a nucleolar marker protein, MoNOP1 fused to red fluorescence protein (RFP), indicating that MoRRP8 is a nucleolar protein. Deletion of the MoRRP8 gene caused a reduction in vegetative growth and impinged largely on asexual sporulation. Although the asexual spores of ΔMorrp8 were morphologically indistinguishable from those of wild-type, they showed delay in germination and reduction in appressorium formation. Our pathogenicity assay revealed that the MoRRP8 is required for full virulence and growth within host plants. Taken together, these results suggest that nucleolar processes mediated by MoRRP8 is pivotal for fungal development and pathogenesis.
... The multiple sequence alignment result suggested the corresponding residues in other reported OMTs were basic residues (His), and residues in NMTs were neutral or acidic (Fig. S22). The LcsG-catalyzed N-methyl transfer reaction is expected to occur via nucleophilic attack by the lone electron pair of terminal N of leucinostatins on the reactive sulfonium methyl group of SAM 43 . The O-methyltransfer reaction needs a base-assisted deprotonation step to generate a nucleophile 44 , and this may be the reason why OMTs share a His residue. ...
N- methyltransferase (NMT)-catalyzed methylations are rarely reported at nonribosomal peptides (NRPs) terminuses. Here, we discovered a fungal NMT LcsG for the iterative terminal N -methyl formation of a family of NRPs, leucinostatins. Gene deletion suggested LcsG is essential to the methylation of leucinostatins. In vitro assay and HRESI-MS-MS analysis proved the methylation sites were the NH 2 , NHCH 3 and N(CH 3 ) 2 in the C-terminal unit of various leucinostatins. Based on the protein structure predicted by artificial intelligence (AI), molecular docking, and site-directed mutagenesis, we proposed the catalytic mechanism of the LcsG-catalyzed reaction was an N atom coordinated by two negatively charged residues (Asp368, Asp395 for LcsG) towards the subsequent S N 2 methylation. These findings not only provide an approach for enriching the variety of natural bioactivity of NPRs but also deepen the insight into the catalytic mechanism of N -methylation of NRPs.
... [1] In nature, only 5'-adenosine triphosphate (ATP) is used more often as an enzyme cosubstrate. [2] The chemical structure of SAM was determined in 1952 by Cantoni et al. and can be divided into two main components: an amino acid part arising from l-methionine, and an adenosyl part derived from ATP. Both moieties are linked by the positively charged sulfonium, activating the cofactor for nucleophilic attack. ...
Methylation reactions are of significant interest when generating pharmaceutically active molecules and building blocks for other applications. Synthetic methylating reagents are often toxic and unselective due to their high reactivity. S‐Adenosyl‐l‐methionine (SAM)‐dependent methyltransferases (MTs) present a chemoselective and environmentally friendly alternative. The anthranilate N‐MT from Ruta graveolens (RgANMT) is involved in acridone alkaloid biosynthesis, methylating anthranilate. Although it is known to methylate substrates only at the N‐position, the closest relatives with respect to amino acid sequence similarities of over 60 % are O‐MTs catalysing the methylation reaction of caffeate and derivatives containing only hydroxyl groups (CaOMTs). In this study, we investigated the substrate range of RgANMT and a CaOMT from Prunus persica (PpCaOMT) using compounds with both, an amino‐ and hydroxyl group (aminophenols) as possible methyl group acceptors. For both enzymes, the reaction was highly chemoselective. Furthermore, generating cofactor derivatives in situ enabled the transfer of other alkyl chains onto the aminophenols, leading to an enlarged pool of products. Selected MT reactions were performed at a preparative biocatalytic scale in in vitro and in vivo experiments resulting in yields of up to 62 %.
... Conversely, the great majority of methyltransferases utilize a common methyl donor, Sadenosyl-methionine (SAM). Methyltransferases are divided into five distinct superfamilies, among which the second largest is the SPOUT (SpoU-TrmD) [3,4]. SPOUT proteins modify mainly RNA, especially rRNA and tRNA [5]. ...
The Nep1 protein is essential for the formation of eukaryotic and archaeal small ribosomal subunits, and it catalyzes the site-directed SAM-dependent methylation of pseudouridine (Ψ) during pre-rRNA processing. It possesses a non–trivial topology, namely, a 31 knot in the active site. Here, we address the issue of seemingly unfeasible deprotonation of Ψ in Nep1 active site by a distant aspartate residue (D101 in S. cerevisiae), using a combination of bioinformatics, computational, and experimental methods. We identified a conserved hydroxyl-containing amino acid (S233 in S. cerevisiae, T198 in A. fulgidus) that may act as a proton-transfer mediator. Molecular dynamics simulations, based on the crystal structure of S. cerevisiae, and on a complex generated by molecular docking in A. fulgidus, confirmed that this amino acid can shuttle protons, however, a water molecule in the active site may also serve this role. Quantum-chemical calculations based on density functional theory and the cluster approach showed that the water-mediated pathway is the most favorable for catalysis. Experimental kinetic and mutational studies reinforce the requirement for the aspartate D101, but not S233. These findings provide insight into the catalytic mechanisms underlying proton transfer over extended distances and comprehensively elucidate the mode of action of Nep1.
... 41 Through sequence homology search followed by biochemical activity validation, nine PRMT members have been discovered consecutively to exist in mammalian cells, all of which belong to the class I of SAM-dependent methyltransferases and bind SAM with a characteristic Rossmann fold. [42][43][44][45][46][47] Based on their end-methylation products, PRMT1, -2, -3, -4, -6, and -8 are grouped into type I enzymes that catalyze methylation of the arginine residue to produce MMA and can further methylate MMA to produce ADMA ( Figure 2). 38,44,[48][49][50][51][52] PRMT4 is also known as coactivator associated arginine methyltransferase 1 (CARM1). ...
Protein arginine methylation is a widespread post-translational modification (PTM) in eukaryotic cells. This chemical modification in proteins functionally modulates diverse cellular processes from signal transduction, gene expression, and DNA damage repair to RNA splicing. The chemistry of arginine methylation entails the transfer of the methyl group from S-adenosyl-l-methionine (AdoMet, SAM) onto a guanidino nitrogen atom of an arginine residue of a target protein. This reaction is catalyzed by about 10 members of protein arginine methyltransferases (PRMTs). With impacts on a variety of cellular processes, aberrant expression and activity of PRMTs have been shown in many disease conditions. Particularly in oncology, PRMTs are commonly overexpressed in many cancerous tissues and positively correlated with tumor initiation, development and progression. As such, targeting PRMTs is increasingly recognized as an appealing therapeutic strategy for new drug discovery. In the past decade, a great deal of research efforts has been invested in illuminating PRMT functions in diseases and developing chemical probes for the mechanistic study of PRMTs in biological systems. In this review, we provide a brief developmental history of arginine methylation along with some key updates in arginine methylation research, with a particular emphasis on the chemical aspects of arginine methylation. We highlight the research endeavors for the development and application of chemical approaches and chemical tools for the study of functions of PRMTs and arginine methylation in regulating biology and disease.
... Methylation to arginine, histidine, and lysine is important for gene expression, regulation of protein activity, and RNA metabolism [323,324]. Methyltransferases catalyze the protein's methylation by using S-adenosyl-L-methionine as a methyl donor [325]. Methylation produces the methylated substrate and S-adenosyl-L-homocysteine, which is then, degraded into adenosine and homocysteine by S-adenosyl-homocysteine hydrolase. ...
The main purpose of this paper was to generate a narrative review related to the current knowledge of the TP53 gene and its product, the p53 protein. It was also attempted to elucidate the different p53 reactivation strategies of great interest, as various small molecules are being studied to reactivate mutant p53. PubMed and ScienceDirect were searched for p53, mutant p53, and wild-type p53 limited by the title filter through the end of 2022. The collected articles were studied, evaluated and summarized. In the short (p) arm of chromosome 17, there is a special place for TP53 . (17p.13.1). It is made up of 19,180 bp, which includes thirteen exons, (elevem exons, two alternative exons), and ten introns. TP53 is mutated in most types of human cancers resulting in aggressive cancer proliferation, immune system evasion, genomic instability, invasion, and metastasis. Under stress-free conditions, p53 function is negatively regulated by HDM2, a p53 target gene, which binds to it and establishes an auto-regulatory negative feedback loop that promotes proteasomal-dependent degradation. In these conditions, p53 maintains at low levels and normalizes biological operations as the master regulator of cell fate. However, under conditions of stress such as DNA damage, hypoxia, oxidative stress, oncogene expression, nutrient deprivation, ribosomal dysfunction, or telomere attrition the p53 selection pathway will be cell type-specific and depend on the type and severity of the cell damage. Post-translational modifications such as phosphorylation and acetylation, which induce the expression of p53 target genes, contribute to the p53 selection pathway. In these conditions, p53 tetramerized and stabilized in the nucleus and activated, and its levels increased in the cell due to blocking the interaction with MDM2. Valuable findings have been discovered that elucidate the biological, biochemical, immunological, physiological, and pathological roles of p53 and its fundamental roles in cancer biology and genetics. The information gathered here should contribute to a better understanding of the impact of p53 deregulation on cancer and new research aimed at finding new anticancer strategies capable of reactivating the cancer suppressive function of WT and/or blocking the function of mutant p53 in order to improve cancer therapy and prognosis.
... It modulates the activity, cellular location, and interactions of proteins. Protein methyltransferases (MTases) are enzymes that catalyze the transfer of a methyl group from the donor S-adenosylmethionine (AdoMet) to a target amino acid residue [1]. The human methyltransferasome contains 208 known or putative MTases, of which most fall in either the seven-β-strand (7BS) family (60% of MTases) or the Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) family (27% of MTases) [2]. ...
The methyltransferase-like protein 13 (METTL13) methylates the eukaryotic elongation factor 1 alpha (eEF1A) on two locations: the N-terminal amino group and lysine 55. The absence of this methylation leads to reduced protein synthesis and cell proliferation in human cancer cells. Previous studies showed that METTL13 is dispensable in non-transformed cells, making it potentially interesting for cancer therapy. However, METTL13 has not been examined yet in whole animals. Here, we used the nematode Caenorhabditis elegans as a simple model to assess the functions of METTL13. Using methyltransferase assays and mass spectrometry, we show that the C. elegans METTL13 (METL-13) methylates eEF1A (EEF-1A) in the same way as the human protein. Crucially, the cancer-promoting role of METL-13 is also conserved and depends on the methylation of EEF-1A, like in human cells. At the same time, METL-13 appears dispensable for animal growth, development, and stress responses. This makes C. elegans a convenient whole-animal model for studying METL13-dependent carcinogenesis without the complications of interfering with essential wild-type functions.
Sinopodophyllum hexandrum ( S. hexandrum ) is an endangered traditional Chinese medicine as abundant podophyllotoxin with powerful anticancer activity. In this study, the rootstalks of S. hexandrum from different geographical locations in China [S1 (Gansu) and S2 (Shaanxi)] were used as research materials to clone the key gene pluviatolide O-methyltransferase 3 ( ShOMT3 ) in the podophyllotoxin biosynthetic pathway. Subsequently, bioinformatics analysis of the ShOMT3 gene and its encoded protein was subjected to bioinformatics analysis using various analysis software including ProtParam, Tmhmm Server 2.0, SubLoc, Signal-P 5.0, and Swiss-model. The results of the analysis revealed that the CDS region of the ShOMT3 gene is 1119 bp long, encoding 372 amino acids. The theoretical molecular weight of the ShOMT3 protein is 41.32784 kD, and the theoretical isoelectric point (pI) is 5.27. The instability coefficient of the protein is 46.05, the aliphatic index is 93.58, and the grand average of hydropathicity (GRAVY) is 0.037, indicating that it is an unstable hydrophobic protein. The protein does not contain transmembrane domains or signal peptides, indicating that it is a non-secreted protein. Secondary structure prediction results suggests that the protein consists of alpha helices, random coils, extended strands, and beta-turns. Tertiary structure prediction results suggests that the protein functions as a monomer. In the phylogenetic tree, the ShOMT3 protein has the highest homology with Podophyllum peltatum ( P. peltatum ). The successful cloning and bioinformatics analysis of the ShOMT3 gene provide theoretical basis and excellent genetic resources for the molecular regulatory mechanism analysis of the podophyllotoxin biosynthetic pathway and molecular breeding in S. hexandrum .
Ribosomal RNA contains many posttranscriptionally modified nucleosides, particularly in the functional parts of the ribosome. The distribution of these modifications varies from one organism to another. In Bacillus subtilis , the model organism for Gram-positive bacteria, mass spectrometry experiments revealed the presence of 7-methylguanosine (m ⁷ G) at position 2574 of the 23S rRNA, which lies in the A-site of the peptidyl transferase center of the large ribosomal subunit. Testing several m ⁷ G methyltransferase candidates allowed to identify the RlmQ enzyme, encoded by the ywbD open reading frame, as the MTase responsible for this modification. The enzyme methylates free RNA and not ribosomal 50S or 70S particles, suggesting that modification occurs in the early steps of ribosome biogenesis
Bisindoles are biologically active natural products that arise from the oxidative dimerization of two molecules of l-tryptophan. In bacterial bisindole pathways, a core set of transformations is followed by the action of diverse tailoring enzymes that catalyze reactions that lead to diverse bisindole products. Among bisindoles, reductasporine is distinct due to its dimethylpyrrolinium structure. Its previously reported biosynthetic gene cluster encodes two unique tailoring enzymes, the imine reductase RedE and the dimethyltransferase RedM, which were shown to produce reductasporine from a common bisindole intermediate in recombinant E. coli. To gain more insight into the unique tailoring enzymes in reductasporine assembly, we reconstituted the biosynthetic pathway to reductasporine in vitro and then solved the 1.7 Å resolution structure of RedM. Our work reveals RedM adopts a variety of conformational changes with distinct open and closed conformations, and site-directed mutagenesis alongside sequence analysis identifies important active site residues. Finally, our work sets the stage for understanding how RedM evolved to react with a pyrrolinium scaffold and may enable the development of new dimethyltransferase catalysts.
Background
Chronic kidney disease (CKD) is a significant global health issue characterized by progressive loss of kidney function. Renal interstitial fibrosis (TIF) is a common feature of CKD, but current treatments are seldom effective in reversing TIF. Nicotinamide N-methyltransferase (NNMT) has been found to increase in kidneys with TIF, but its role in renal fibrosis is unclear.
Methods
Using mice with unilateral ureteral obstruction (UUO) and cultured renal interstitial fibroblast cells (NRK-49F) stimulated with transforming growth factor-β1 (TGF-β1), we investigated the function of NNMT in vivo and in vitro .
Results
We performed single-cell transcriptome sequencing (scRNA-seq) on the kidneys of mice and found that NNMT increased mainly in fibroblasts of UUO mice compared to sham mice. Additionally, NNMT was positively correlated with the expression of renal fibrosis-related genes after UUO injury. Knocking down NNMT expression reduced fibroblast activation and was accompanied by an increase in DNA methylation of p53 and a decrease in its phosphorylation.
Conclusions
Our findings suggest that chronic kidney injury leads to an accumulation of NNMT, which might decrease p53 methylation, and increase the expression and activity of p53. We propose that NNMT promotes fibroblast activation and renal fibrosis, making NNMT a novel target for preventing and treating renal fibrosis.
The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.
Germ cells differentiate into oocytes that launch the next generation upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrently with oocyte specification. Here, we discovered that nuclear SETDB1 is required for silencing a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for upregulating 18 of the ∼30 nucleoporins (Nups) that compose the nucleopore complex (NPC), promoting NPC formation. NPCs anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to the loss of SETDB1 or Nups results in the loss of oocyte identity, cell death, and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification, which is critical for establishing oocyte identity.
In biological systems, proteins can bind to nanoparticles to form a “corona” of adsorbed molecules. The nanoparticle corona is of high interest because it impacts the organism’s response to the nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. Ultimately, a residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Through lysine methylation, we observe subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX measurements reveal that certain regions of the R2ab protein undergo faster exchange rates in the presence of PSNPs, suggesting conformational changes upon binding. Both results support a recently proposed “adsorbotope” model, wherein adsorbed proteins consist of unfolded anchor points interspersed with regions of partial structure. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how proteins respond to nanoparticle surfaces, this research emphasizes the need for advanced methods to comprehend these intricate interactions fully at the residue level.
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Lysine methylation and hydrogen-deuterium exchange can reveal useful structural details about protein adsorption to nanoparticle surfaces.
Lung squamous cell carcinoma (LUSC) is a non-small cell lung cancer with a poor prognosis owing to late diagnosis. New molecular markers are urgently needed to improve the diagnosis and prognosis of LUSC. 7-Methylguanosine (m7G) modifications-a transfer RNA (tRNA) modification-are common in eubacteria, eukaryotes, and a few archaea. These modifications promote the turnover and stability of some mRNAs to prevent mRNA decay, improve translation efficiency, and reduce ribosomal pausing but are associated with poor survival in human cancer cells. However, the expression of m7G-related genes in LUSC and their association with prognosis remain unclear. In this study, we identified nine differentially expressed genes related to prognosis by comparing the expression profiles of tumor tissues (502 LUSC reports) with normal tissues (49 adjacent non-tumor lung tissue reports). The genes included six upregulated (KLK7, LCE3E, AREG, KLK6, ZBED2, and MAPK4) and three downregulated (ADH1C, NTS, and ERLIN2) genes. Based on these nine genes, patients with LUSC were classified into low- and high-risk groups to analyze the trends in prognosis. We found that the nine m7G-related genes play important roles in immune regulation, hormone regulation, and drug sensitivity through pathways, including antigen processing and presentation, adherent plaques, ECM receptor interactions, the drug metabolism of cytochrome p450, and the metabolism of cytochrome p450 to xenobiotics; the functions of these genes are likely accomplished in part by m6A modifications. The effect of m7G-related genes on the diagnosis and prognosis of LUSC was further indicated by population analysis.
Less studied than the other protein arginine methyltransferase isoforms, PRMT7 and PRMT9 have recently been identified as important therapeutic targets. Yet, most of their biological roles and functions are still to be defined, as well as the structural requirements that could drive the identification of selective modulators of their activity. We recently described the structural requirements that led to the identification of potent and selective PRMT4 inhibitors spanning both the substrate and the cosubstrate pockets. The reanalysis of the data suggested a PRMT7 preferential binding for shorter derivatives and prompted us to extend these structural studies to PRMT9. Here, we report the identification of the first potent PRMT7/9 inhibitor and its binding mode to the two PRMT enzymes. Label-free quantification mass spectrometry confirmed significant inhibition of PRMT activity in cells. We also report the setup of an effective AlphaLISA assay to screen small molecule inhibitors of PRMT9.
Methylation reactions are of significant interest when generating pharmaceutically active molecules and building blocks for other applications. Synthetic methylating reagents are often toxic and unselective due to their high reactivity. S -Adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) present a chemoselective and environmentally friendly alternative. The anthranilate N -MT from Ruta graveolens ( Rg ANMT) is involved in acridone alkaloid biosynthesis, methylating anthranilate. Although it is known to methylate substrates only at the N- position, the closest relatives with respect to amino acid sequence similarities of over 60% are O -MTs catalysing the methylation reaction of caffeate and derivatives containing only hydroxyl groups (CaOMTs). In this study, we investigated the substrate range of Rg ANMT and a CaOMT from Prunus persica ( Pp CaOMT) using compounds with both, an amino- and hydroxyl group (aminophenols) as possible methyl group acceptors. For both enzymes, the reaction was highly chemoselective. Furthermore, generating cofactor derivatives in situ enabled the transfer of other alkyl chains onto the aminophenols, leading to an enlarged pool of products. Selected MT reactions were performed at a preparative biocatalytic scale in in vitro and in vivo experiments resulting in yields of up to 62%.
Methylation of DNA plays a key role in diverse biological processes spanning from bacteria to mammals. DNA methyltransferases (MTases) typically employ S-adenosyl-l-methionine (SAM) as a critical cosubstrate and the relevant methyl donor for modification of the C5 position of cytosine. Recently, work on the CpG-specific bacterial MTase, M.MpeI, has shown that a single N374K point mutation can confer the enzyme with the neomorphic ability to use the sparse, naturally occurring metabolite carboxy-S-adenosyl-l-methionine (CxSAM) in order to generate the unnatural DNA modification, 5-carboxymethylcytosine (5cxmC). Here, we aimed to investigate the mechanistic basis for this DNA carboxymethyltransferase (CxMTase) activity by employing a combination of computational modeling and in vitro characterization. Modeling of substrate interactions with the enzyme variant allowed us to identify a favorable salt bridge between CxSAM and N374K that helps to rationalize selectivity of the CxMTase. Unexpectedly, we also discovered a potential role for a key active site E45 residue that makes a bidentate interaction with the ribosyl sugar of CxSAM, located on the opposite face of the CxMTase active site. Prompted by these modeling results, we further explored the space-opening E45D mutation and found that the E45D/N374K double mutant in fact inverts selectivity, preferring CxSAM over SAM in biochemical assays. These findings provide new insight into CxMTase active site architecture and may offer broader utility given the numerous opportunities offered by using SAM analogs for selective molecular labeling in concert with nucleic acid or even protein-modifying MTases.
Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI are described. With poly(dG-dC) as substrate, the reaction proceeds by an equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and its generation during reactions results in non-linear kinetics. AdoMet and AdoHcy significantly interact with only the substrate enzyme-DNA complex; they do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex. In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of substrate cytosines for protons of water at about 7-fold the rate of methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and reassociation of the enzyme with other substrate sequences. Our studies reveal that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves attack of the C6 of substrate cytosines by an enzyme nucleophile and formation of a transient covalent adduct. Based on precedents of other enzymes which catalyze similar reactions and the susceptibility of HhaI to inactivation by N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have been determined to date and is found in related enzymes. Finally, we discuss the possibility that covalent adducts between C6 of pyrimidines and nucleophiles of proteins may be important general components of protein-nucleic acid interactions.
A novel S-adenosyl- L-methionine:halide/bisulfide methyltransferase (EC 2.1.1.-) was purified approximately 1000-fold to apparent homogeneity from leaves of Brassica oleracea. The enzyme catalyzed the S-adenosyl- L-methionine-dependent methylation of the halides iodide, bromide, and chloride to monohalomethanes and of bisulfide to methanethiol. The dual function of the enzyme was demonstrated through co-purification of the halide- and bisulfide-methylating activities in the same ratio and by studies of competition between the alternative substrates iodide and bisulfide. The purification procedure included gel filtration, anion exchange chromatography, and affinity chromatography on adenosine-agarose. Elution of the protein from a chromatofocusing column indicated a pI value of 4.8. The pH optimum of halide methylation (5.5-7.0) was different from that of bisulfide methylation (7.0-8.0). The molecular mass values for the native and denatured protein were 29.5 and 28 kDa, respectively, suggesting that the active enzyme is a monomer. The enzyme had the highest specificity constant for iodide and the next highest for bisulfide. Substrate interaction kinetics and product inhibition patterns were consistent with an Ordered Bi Bi mechanism.
It is known that the same reaction may be catalyzed by structurally unrelated enzymes. We performed a systematic search for such analogous (as opposed to homologous) enzymes by evaluating sequence conservation among enzymes with the same enzyme classification (EC) number using sensitive, iterative sequence database search methods. Enzymes without detectable sequence similarity to each other were found for 105 EC numbers (a total of 243 distinct proteins). In 34 cases, independent evolutionary origin of the suspected analogous enzymes was corroborated by showing that they possess different structural folds. Analogous enzymes were found in each class of enzymes, but their overall distribution on the map of biochemical pathways is patchy, suggesting multiple events of gene transfer and selective loss in evolution, rather than acquisition of entire pathways catalyzed by a set of unrelated enzymes. Recruitment of enzymes that catalyze a similar but distinct reaction seems to be a major scenario for the evolution of analogous enzymes, which should be taken into account for functional annotation of genomes. For many analogous enzymes, the bacterial form of the enzyme is different from the eukaryotic one; such enzymes may be promising targets for the development of new antibacterial drugs.
Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI are described. With poly(dG-dC) as substrate, the reaction proceeds by an equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and its generation during reactions results in non-linear kinetics. AdoMet and AdoHcy significantly interact with only the substrate enzyme-DNA complex; they do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex. In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of substrate cytosines for protons of water at about 7-fold the rate of methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and reassociation of the enzyme with other substrate sequences. Our studies reveal that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves attack of the C6 of substrate cytosines by an enzyme nucleophile and formation of a transient covalent adduct. Based on precedents of other enzymes which catalyze similar reactions and the susceptibility of HhaI to inactivation by N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have been determined to date and is found in related enzymes. Finally, we discuss the possibility that covalent adducts between C6 of pyrimidines and nucleophiles of proteins may be important general components of protein-nucleic acid interactions.
Selectivity of catechol O-methyltransferase has been examined for the three ring-fluorinated norepinephrines to elucidate the role of acidity of the phenolic groups in their methylation. Substitution of fluorine at the 5-position of norepinephrine reverses the selectivity of catechol O-methyltransferase so that p-O-methylation predominates. The 5-fluoro substituent also causes the pKa of the p-hydroxyl group to decrease substantially. In contrast, 2- and 6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group. These results suggest that acidity of a phenolic group can play an important role in its ability to be methylated by catechol O-methyltransferase. Percentages of p-O-methylation of norepinephrine and its fluorinated derivatives increase with pH. This relative increase in p-O-methylation appears to accompany ionization of a group with pKa of 8.6, 7.7, 7.9, and 8.4 for norepinephrine and its 2-, 5-, and 6-fluoro derivative, respectively. These pKa values are the same as or similar to the pKa values of a phenolic hydroxyl group of these substrates. 3,4-Dihydroxybenzyl alcohol and its 5-fluoro derivative are O-methylated by catechol O-methyltransferase to form p- and m-O-methyl products in approximately 1:1 and 4:1 ratios, respectively, at all pH values. Based on the above results, a catechol-binding site model for catechol O-methyltransferase is proposed in which the two phenolic hydroxyl groups of catechol substrates are postulated to be approximately equally spaced from the methyl group of the cosubstrate S-adenosylmethionine.
The steric course of the methyl group transfer catalyzed by catechol O-methyltransferase was studied using S-adenosylmethionine (AdoMet) carrying a methyl group made chiral by labeling with 1H, 2H, and 3H in an asymmetrical arrangement. Incubation of the two diastereomers of this substrate with catechol O-methyl-transferase purified from rat liver and epinephrine or protocatechuic acid as acceptor gave the corresponding methylated catechols. These were degraded to convert the methoxy group in a series of stereochemically unambiguous reactions into the methyl group of acetate, which was then analyzed for its configuration. The results indicate that the transfer of the methyl group from AdoMet to either acceptor occurs in an inversion mode. The catechol O-methyltransferase reaction thus involves a direct transfer of the methyl group from the sulfur of AdoMet to the oxygen of the catechol in an SN2 process, without a methylated enzyme intermediate.
4′-Thio-2′-deoxycytidine was synthesized as a 5′-protected phosphoramidite compatible with solid phase DNA synthesis. When
incorporated as the target cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M.HhaI, methyl transfer was strongly inhibited. In contrast, these same oligonucleotides were normal substrates for the cognate
restriction endonuclease R.HhaI and its isoschizomer R.HinP1I. M.HhaI was able to bind both 4′-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium conditions. However,
the presence of 4′-thio-2′-deoxycytidine decreased the half-life of the complex by >10-fold. The crystal structure of a ternary
complex of M.HhaI, AdoMet and DNA containing 4′-thio-2′-deoxycytidine was solved at 2.05 Å resolution with a crystallographic R-factor of
0.186 and R-free of 0.231. The structure is not grossly different from previously solved ternary complexes containing M.HhaI, DNA and AdoHcy. The difference electron density suggests partial methylation at C5 of the flipped target 4′-thio-2′-deoxycytidine.
The inhibitory effect of the 4′ sulfur atom on enzymatic activity may be traced to perturbation of a step in the methylation
reaction after DNA binding but prior to methyl transfer. This inhibitory effect can be partially overcome after a considerably
long time in the crystal environment where the packing prevents complex dissociation and the target is accurately positioned
within the active site.
We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.
Biosynthesis of the corrin ring of vitamin B12 requires the action of six S-adenosyl-L-methionine (AdoMet) dependent transmethylases, closely related in sequence. The first X-ray structure of one of these, cobalt-precorrin-4 transmethylase, CbiF, from Bacillus megaterium has been determined to a resolution of 2.4 A. CbiF contains two alphabeta domains forming a trough in which S-adenosyl-L-homocysteine (AdoHcy) binds. The location of AdoHcy and a number of conserved residues, helps define the precorrin binding site. A second crystal form determined at 3.1 A resolution highlights the flexibility of two loops around this site. CbiF employs a unique mode of AdoHcy binding and represents a new class of transmethylase.
The Escherichia coli fmugene product has recently been determined to be the 16S rRNA m5C 967 methyl-transferase. As such, Fmu represents the first protein identified as an S-adenosyl-L-methionine (AdoMet)-dependent RNA m5C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu as an initial probe in an iterative
search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m5C methyltransferases. Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more
than 30 additional homologs. These putative RNA m5C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts,
or orthologs. The enzymes proposed to be RNA m5C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m5C methyltransferases and other AdoMet-dependent methyltransferases. Structure-function correlates in the known AdoMet methyltransferases
support the assignment of this family as RNA m5C methyltransferases.
DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction–modification
systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction
endonucleases afforded by the bacterial methyltransferases. The first structure of an N6-adenine methyltransferase belonging to the β class of bacterial methyltransferases is described here. The structure of M·RsrI from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 Å resolution using X-ray crystallography.
Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The
catalytic core most closely resembles the PvuII methyltransferase, a cytosine amino methyltransferase of the same β group. The larger nucleotide binding pocket observed
in M·RsrI is expected because it methylates adenine. However, the most striking difference between the RsrI methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which
is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This
observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.
The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.
Chalcone O-methyltransferase (ChOMT) and isoflavone O-methyltransferase (IOMT) are S-adenosyl-l-methionine (SAM) dependent plant natural product methyltransferases involved in secondary metabolism in Medicago sativa (alfalfa). Here we report the crystal structure of ChOMT in complex with the product S-adenosyl-l-homocysteine and the substrate isoliquiritigenin (4,2',4'-trihydroxychalcone) refined to 1.8 A as well as the crystal structure of IOMT in complex with the products S-adenosyl-l-homocysteine and isoformononetin (4'-hydroxy-7-methoxyisoflavone) refined to 1.4 A. These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection. As such, this work provides a structural basis for understanding the substrate specificity of the diverse family of plant OMTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.
Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow 'trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep 'figure-of-eight' knot which had not been visually identified. In the present study, the crystal structure of a hypothetical RNA 2'-O-ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 A resolution and a deep trefoil knot was found for the first time. The present knot is formed by the threading of a 44-residue polypeptide chain through a 41-residue loop and is better defined than the previously reported knots. Two of the three catalytic residues conserved in the 2'-O-ribose methyltransferase family are located in the knotting loop and in the knotted carboxy-terminal chain, which is the first observation that the enzyme active site is constructed right on the knot. On the other hand, the amino-terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA.
MT-A70 is the S-adenosylmethionine-binding subunit of human mRNA:m(6)A methyl-transferase (MTase), an enzyme that sequence-specifically methylates adenines in pre-mRNAs. The physiological importance yet limited understanding of MT-A70 and its apparent lack of similarity to other known RNA MTases combined to make this protein an attractive target for bioinformatic analysis. The sequence of MT-A70 was subjected to extensive in silico analysis to identify orthologous and paralogous polypeptides. This analysis revealed that the MT-A70 family comprises four subfamilies with varying degrees of interrelatedness. One subfamily is a small group of bacterial DNA:m(6)A MTases. The other three subfamilies are paralogous eukaryotic lineages, two of which have not been associated with MTase activity but include proteins having substantial regulatory effects. Multiple sequence alignments and structure prediction for members of all four subfamilies indicated a high probability that a consensus MTase fold domain is present. Significantly, this consensus fold shows the permuted topology characteristic of the b class of MTases, which to date has only been known to include DNA MTases.
We have determined the structure of PvuII methyltransferase (M.PvuII) complexed with S-adenosyl-l-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethioninesubstituted protein.
M.PvuII catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its
recognition sequence 5′-CAGCTG-3′. The protein is dominated by an open α/β-sheet structure with a prominent V-shaped cleft:
AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent
with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases
that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M.PvuII α/β-sheet structure is very similar to those of M.HhaI (a cytosine C5 methyltransferase) and M.TaqI (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural
fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common
fold is a seven-stranded β-sheet (6↓ 7↑ 5↓ 4↓ 1↓ 2↓ 3↓) formed by five parallel β-strands and an antiparallel β-hairpin. The
β-sheet is flanked by six parallel α-helices, three on each side. The AdoMet binding site is located at the C-terminal ends
of strands β1 and β2 and the active site is at the C-terminal ends of strands β4 and β5 and the N-terminal end of strand β7.
The AdoMet-protein interactions are almost identical among M.PvuII, M.HhaI and M.TaqI, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among
the active sites of M.PvuII, M.TaqI and M.HhaI reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for
cytosine C5 methylation, suggesting a mechanism for amino methylation.
The S-adenosylmethionine-dependent methyltransferase enzymes share little sequence identity, but incorporate a highly conserved structural fold. Surprisingly, residues that bind the common cofactor are poorly conserved, although the binding site is localised to the same region of the fold. The substrate-binding region of the fold varies enormously. Over the past two years, there has been a significant increase in the number of structures that are known to incorporate this fold, including several uncharacterized proteins and two proteins that lack methyltransferase activity.
Background: Formation of isoaspartyl residues is one of several processes that damage proteins as they age. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) is a conserved and nearly ubiquitous enzyme that catalyzes the repair of proteins damaged by isoaspartyl formation.Results: We have determined the first structure of a PIMT from crystals of the T. maritima enzyme complexed to S-adenosyl-L-homocysteine (AdoHcy) and refined it to 1.8 Å resolution. Although PIMT forms one structural unit, the protein can be divided functionally into three subdomains. The central subdomain closely resembles other S-adenosyl-L-methionine-dependent methyltransferases but bears a striking alteration of topological connectivity, which is not shared by any other member of this family. Rather than arranged as a mixed β sheet with topology 6↑7↓5↑4↑1↑2↑3↑, the central sheet of PIMT is reorganized to 7↑6↓5↑4↑1↑2↑3↑. AdoHcy is largely buried between the N-terminal and central subdomains by a conserved and largely hydrophobic loop on one rim of the binding cleft, and a conserved Ser/Thr-rich β strand on the other. The Ser/Thr-rich strand may provide hydrogen bonds for specific interactions with isoaspartyl substrates. The side chain of Ile-206, a conserved residue, crosses the cleft, restricting access to the donor methyl group to a deep well, the putative isoaspartyl methyl acceptor site.Conclusions: The structure of PIMT reveals a unique modification of the methyltransferase fold along with a site for specific recognition of isoaspartyl substrates. The sequence conservation among PIMTs suggests that the current structure should prove a reliable model for understanding the repair of isoaspartyl damage in all organisms.
The first three-dimensional structure of a DNA methyltransferase is presented. The crystal structure of the DNA (cytosine-5)-methyltransferase, M.HhaI (recognition sequence: GCGC), complexed with S-adenosyl-L-methionine has been determined and refined at 2.5 A resolution. The core of the structure is dominated by sequence motifs conserved among all DNA (cytosine-5)-methyltransferases, and these are responsible for cofactor binding and methyltransferase function.
The reaction mechanism of the nonenzymatic transmethylation of catechol by S-adenosylmethionine (AdoMet, as modeled by sulfonium ion) has been elucidated using ab initio and semiempirical quantum mechanical methods. The gas phase reaction between catecholate and sulfonium is extremely fast, involving no overall barrier. The reaction profile to some extent resembles a typical gas phase S N 2 reaction. However, in aqueous solution, this reaction is very slow with a predicted barrier of 37.3 kcal/mol. The calculated (k H /k D) R , k 12 /k 13 , k 16 /k 18 , and k 32 /k 34 are 0.80, 1.06, 1.003, and 1.010, respectively. Previously, Schowen and co-workers measured (k H /k D) R and k 12 /k 13 to be 0.83 (0.05 and 1.09 (0.05 for the catechol O-methyltransferase (COMT)-catalyzed methylation of 3,4-dihydroxyacetophenone by AdoMet. This good agreement between the calculated kinetic isotope effects for the model reaction and the measured kinetic isotope effects for the enzymatic reaction seems to suggest that the structure of the enzymatic transition state is very similar to that of the nonenzymatic reaction. Factors that modulate the catalytic efficacy of catechol O-methyltransferase were discussed in light of the present study on the nonenzymatic reaction and the recently solved X-ray crystal structure.
A series of Ba3ZnZCo2-ZFe24O41/SiO2 microcrystalline glass ceramics with Z=0.0,0.4,0.8 and 1.2 were prepared by citrate sol-gel process. The result showed that
Ba3ZnZCo2-ZFe24O41 hexferrite crystallites could be obtained by this process at 1200 °C in the system of BaO-Fe2O3-CoO-ZnO-SiO2. The complex dielectric constant and complex permeability of Ba3ZnZCo2-ZFe24O41/SiO2 microcrystalline glass ceramics calcined at different temperature were measured in the range of 200MHz-6 GHz by transmission/reflection
coaxial line method. The complex dielectric constant and dielectric loss exhibited insignificant variety in the whole range
of measuring frequencies for all samples. The real part of the permeability decreased as the measuring frequency increasing,
and calcining temperature had a clear influence on the value of µ′ for Ba3ZnZCo2-ZFe24O41/SiO2 microcrystalline glass ceramics, and so did the content of Zn2+ and Co2+. The natural resonance phenomenon was observed in µ′′ spectra for all the Ba3ZnZCo2-ZFe24O41/SiO2 microcrystalline glass ceramics. The substitution of Zn2+ ion and annealing temperature closely affect the resonance frequency, the more the Zn2+ ion, the higher the annealing temperature and the lower the resonance frequency.
Glycine N-methyltransferase (S-adenosyl-l-methionine: glycine methyltransferase, EC 2.1.1.20; GNMT) catalyzes the AdoMet-dependent methylation of glycine to form sarcosine (N-methylglycine). Unlike most methyltransferases, GNMT is a tetrameric protein showing a positive cooperativity in AdoMet binding and weak inhibition by S-adenosylhomocysteine (AdoHcy). The first crystal structure of GNMT complexed with AdoMet showed a unique “closed” molecular basket structure, in which the N-terminal section penetrates and corks the entrance of the adjacent subunit. Thus, the apparent entrance or exit of the active site is not recognizable in the subunit structure, suggesting that the enzyme must possess a second, enzymatically active, “open” structural conformation. A new crystalline form of the R175K enzyme has been grown in the presence of an excess of AdoHcy, and its crystal structure has been determined at 3.0 Å resolution. In this structure, the N-terminal domain (40 amino acid residues) of each subunit has moved out of the active site of the adjacent subunit, and the entrances of the active sites are now opened widely. An AdoHcy molecule has entered the site occupied in the “closed” structure by Glu15 and Gly16 of the N-terminal domain of the adjacent subunit. An AdoHcy binds to the consensus AdoMet binding site observed in the other methyltransferase. This AdoHcy binding site supports the glycine binding site (Arg175) deduced from a chemical modification study and site-directed mutagenesis (R175K). The crystal structures of WT and R175K enzymes were also determined at 2.5 Å resolution. These enzyme structures have a closed molecular basket structure and are isomorphous to the previously determined AdoMet-GNMT structure. By comparing the open structure to the closed structure, mechanisms for auto-inhibition and for the forced release of the product AdoHcy have been revealed in the GNMT structure. The N-terminal section of the adjacent subunit occupies the AdoMet binding site and thus inhibits the methyltransfer reaction, whereas the same N-terminal section forces the departure of the potentially potent inhibitor AdoHcy from the active site and thus facilitates the methyltransfer reaction. Consequently GNMT is less active at a low level of AdoMet concentration, and is only weakly inhibited by AdoHcy. These properties of GNMT are particularly suited for regulation of the cellular AdoMet/AdoHcy ratio.
Protein l-isoaspartyl (d-aspartyl) methyltransferases (EC 2.1.1.77) are found in almost all organisms. These enzymes catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of isomerized and racemized aspartyl residues in age-damaged proteins as part of an essential protein repair process. Here, we report crystal structures of the repair methyltransferase at resolutions up to 1.2 Å from the hyperthermophilic archaeon Pyrococcus furiosus. Refined structures include binary complexes with the active cofactor AdoMet, its reaction product S-adenosylhomocysteine (AdoHcy), and adenosine. The enzyme places the methyl-donating cofactor in a deep, electrostatically negative pocket that is shielded from solvent. Across the multiple crystal structures visualized, the presence or absence of the methyl group on the cofactor correlates with a significant conformational change in the enzyme in a loop bordering the active site, suggesting a role for motion in catalysis or cofactor exchange. We also report the structure of a ternary complex of the enzyme with adenosine and the methyl-accepting polypeptide substrate VYP(l-isoAsp)HA at 2.1 Å. The substrate binds in a narrow active site cleft with three of its residues in an extended conformation, suggesting that damaged proteins may be locally denatured during the repair process in cells. Manual and computer-based docking studies on different isomers help explain how the enzyme uses steric effects to make the critical distinction between normal l-aspartyl and age-damaged l-isoaspartyl and d-aspartyl residues.
Methionine synthase (MetH) from Escherichia coli catalyzes the synthesis of methionine from homocysteine and methyltetrahydrofolate via two methyl transfer reactions that are mediated by the endogenous cobalamin cofactor. After binding both substrates in a ternary complex, the enzyme transfers a methyl group from the methylcobalamin cofactor to homocysteine, generating cob(I)alamin enzyme and methionine. The enzyme then catalyzes methyl transfer from methyltetrahydrofolate to the cob(I)alamin cofactor, forming methylcobalamin cofactor and tetrahydrofolate prior to the release of both products. The cob(I)alamin form of the enzyme occasionally undergoes oxidation to an inactive cob(II)alamin species; the enzyme also catalyzes its own reactivation. Electron transfer from reduced flavodoxin to the cob(II)alamin cofactor is thought to generate cob(I)alamin enzyme, which is then trapped by methyl transfer from adenosylmethionine to the cobalt, restoring the enzyme to the active methylcobalamin form. Thus the enzyme is potentially able to catalyze two methyl transfers to the cob(I)alamin cofactor: methyl transfer from methyltetrahydrofolate during primary turnover and methyl transfer from adenosylmethionine during activation. It has recently been shown that methionine synthase is constructed from at least four separable regions that are responsible for binding each of the three substrates and the cobalamin cofactor, and it has been proposed that changes in positioning of the substrate binding regions vis-à-vis the cobalamin binding region could allow the enzyme to control which substrate has access to the cofactor. In this paper, we offer evidence that methionine synthase exists in two different conformations that interconvert in the cob(II)alamin oxidation state. In the primary turnover conformation, the enzyme reacts with homocysteine and methyltetrahydrofolate but is unreactive toward adenosylmethionine and flavodoxin. In the reactivation conformation, the enzyme is active toward adenosylmethionine and flavodoxin but unreactive toward methyltetrahydrofolate. The two conformations differ in the susceptibility of the substrate-binding regions to tryptic proteolysis. We propose a model in which conformational changes control access to the cobalamin cofactor and are the primary means of controlling cobalamin reactivity in methionine synthase.
Previous X-ray crystallographic studies have revealed that the catalytic domain of a DNA methyltransferase (Mtase) generating C5-methylcytosine bears a striking structural similarity to that of a Mtase generating N6-methyladenine. Guided by this common structure, we performed a multiple sequence alignment of 42 amino-Mtases (N6-adenine and N4-cytosine). This comparison revealed nine conserved motifs, corresponding to the motifs I to VIII and X previously defined in C5-cytosine Mtases. The amino and C5-cytosine Mtases thus appear to be more closely related than has been appreciated. The amino Mtases could be divided into three groups, based on the sequential order of motifs, and this variation in order may explain why only two motifs were previously recognized in the amino Mtases. The Mtases grouped in this way show several other group-specific properties, including differences in amino acid sequence, molecular mass and DNA sequence specificity. Surprisingly, the N4-cytosine and N6-adenine Mtases do not form separate groups. These results have implications for the catalytic mechanisms, evolution and diversification of this family of enzymes. Furthermore, a comparative analysis of the S-adenosyl-L-methionine and adenine/cytosine binding pockets suggests that, structurally and functionally, they are remarkably similar to one another.
A novel S-adenosyl-L-methionine:halide/bisulfide methyltransferase (EC 2.1.1.-) was purified approximately 1000-fold to apparent homogeneity from leaves of Brassica oleracea. The enzyme catalyzed the S-adenosyl-L-methionine-dependent methylation of the halides iodide, bromide, and chloride to monohalomethanes and of bisulfide to methanethiol. The dual function of the enzyme was demonstrated through co-purification of the halide- and bisulfide-methylating activities in the same ratio and by studies of competition between the alternative substrates iodide and bisulfide. The purification procedure included gel filtration, anion exchange chromatography, and affinity chromatography on adenosine-agarose. Elution of the protein from a chromatofocusing column indicated a pI value of 4.8. The pH optimum of halide methylation (5.5-7.0) was different from that of bisulfide methylation (7.0-8.0). The molecular mass values for the native and denatured protein were 29.5 and 28 kDa, respectively, suggesting that the active enzyme is a monomer. The enzyme had the highest specificity constant for iodide and the next highest for bisulfide. Substrate interaction kinetics and product inhibition patterns were consistent with an Ordered Bi Bi mechanism.
Catechol O-methyltransferase (COMT, EC 2.1.1.6) is important in the central nervous system because it metabolizes catecholamine neurotransmitters such as dopamine. The enzyme catalyses the transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to one hydroxyl group of catechols. COMT also inactivates catechol-type compounds such as L-DOPA. With selective inhibitors of COMT in combination with L-DOPA, a new principle has been realized in the therapy of Parkinson's disease. Here we solve the atomic structure of COMT to 2.0 A resolution, which provides new insights into the mechanism of the methyl transfer reaction. The co-enzyme-binding domain is strikingly similar to that of an AdoMet-dependent DNA methylase, indicating that all AdoMet methylases may have a common structure.
The crystal structure has been determined at 2.8 A resolution for a chemically-trapped covalent reaction intermediate between the HhaI DNA cytosine-5-methyltransferase, S-adenosyl-L-homocysteine, and a duplex 13-mer DNA oligonucleotide containing methylated 5-fluorocytosine at its target. The DNA is located in a cleft between the two domains of the protein and has the characteristic conformation of B-form DNA, except for a disrupted G-C base pair that contains the target cytosine. The cytosine residue has swung completely out of the DNA helix and is positioned in the active site, which itself has undergone a large conformational change. The DNA is contacted from both the major and the minor grooves, but almost all base-specific interactions between the enzyme and the recognition bases occur in the major groove, through two glycine-rich loops from the small domain. The structure suggests how the active nucleophile reaches its target, directly supports the proposed mechanism for cytosine-5 DNA methylation, and illustrates a novel mode of sequence-specific DNA recognition.
The first three-dimensional structure of a DNA methyltransferase is presented. The crystal structure of the DNA (cytosine-5)-methyltransferase, M.HhaI (recognition sequence: GCGC), complexed with S-adenosyl-L-methionine has been determined and refined at 2.5 A resolution. The core of the structure is dominated by sequence motifs conserved among all DNA (cytosine-5)-methyltransferases, and these are responsible for cofactor binding and methyltransferase function.
Background:
In both mammalian and microbial species, B12-dependent methionine synthase catalyzes methyl transfer from methyltetrahydrofolate (CH3-H4folate) to homocysteine. The B12 (cobalamin) cofactor plays an essential role in this reaction, accepting the methyl group from CH3-H4folate to form methylcob(III)alamin and in turn donating the methyl group to homocysteine to generate methionine and cob(I)alamin. Occasionally the highly reactive cob(I)alamin intermediate is oxidized to the catalytically inactive cob(II)alamin form. Reactivation to sustain enzyme activity is achieved by a reductive methylation, requiring S-adenosylmethionine (AdoMet) as the methyl donor and, in Esherichia coli, flavodoxin as an electron donor. The intact system is controlled and organized so that AdoMet, rather than methyltetrahydrofolate, is the methyl donor in the reactivation reaction. AdoMet is not wasted as a methyl donor in the catalytic cycle in which methionine is synthesized from homocysteine. The structures of the AdoMet binding site and the cobalamin-binding domains (previously determined) provide a starting point for understanding the methyl transfer reactions of methionine synthase.
Results:
We report the crystal structure of the 38 kDa C-terminal fragment of E.coli methionine synthase that comprises the AdoMet-binding site and is essential for reactivation. The structure, which includes residues 901-1227 of methionine synthase, is a C-shaped single domain whose central feature is a bent antiparallel betasheet. Database searches indicate that the observed polypeptide has no close relatives. AdoMet binds near the center of the inner surface of the domain and is held in place by both side chain and backbone interactions.
Conclusions:
The conformation of bound AdoMet, and the interactions that determine its binding, differ from those found in other AdoMet-dependent enzymes. The sequence Arg-x-x-x-Gly-Tyr is critical for the binding of AdoMet to methionine synthase. The position of bound AdoMet suggests that large areas of the C-terminal and cobalamin-binding fragments must come in contact in order to transfer the methyl group of AdoMet to cobalamin. The catalytic and activation cycles may be turned off and on by alternating physical separation and approach of the reactants.
Background:
Flagellated bacteria swim towards favorable chemicals and away from deleterious ones. The sensing of chemoeffector gradients involves chemotaxis receptors, transmembrane proteins that detect stimuli through their periplasmic domains and transduce signals via their cytoplasmic domains to the downstream signaling components. Signaling outputs from chemotaxis receptors are influenced both by the binding of the chemoeffector ligand to the periplasmic domain and by methylation of specific glutamate residues on the cytoplasmic domain of the receptor. Methylation is catalyzed by CheR, an S-adenosylmethionine-dependent methyltransferase. CheR forms a tight complex with the receptor by binding a region of the receptors that is distinct from the methylation site. CheR belongs to a broad class of enzymes involved in the methylation of a variety of substrates. Until now, no structure from the class of protein methyltransferases has been characterized.
Results:
The structure of the Salmonella typhimurium chemotaxis receptor methyltransferase CheR bound to S-adenosylhomocysteine, a product and inhibitor of the methylation reaction, has been determined at 2.0 A resolution. The structure reveals CheR to be a two-domain protein, with a smaller N-terminal helical domain linked through a single polypeptide connection to a larger C-terminal alpha/beta domain. The C-terminal domain has the characteristics of a nucleotide-binding fold, with an insertion of a small antiparallel beta sheet subdomain. The S-adenosylhomocysteine-binding site is formed mainly by the large domain, with contributions from residues within the N-terminal domain and the linker region.
Conclusions:
The CheR structure shares some structural similarities with small molecule DNA and RNA methyltransferases, despite a lack of sequence similarity among them. In particular, there is significant structural preservation of the S-adenosylmethionine-binding clefts; the specific length and conformation of a loop in the alpha/beta domain seems to be required for S-adenosylmethionine binding within these enzymes. Unique structural features of CheR, such as the beta subdomain, are probably necessary for CheR's specific interaction with its substrates, the bacterial chemotaxis receptors.
Several contemporary enzymes catalyze alternative reactions distinct from their normal biological reactions. In some cases the alternative reaction is similar to a reaction that is efficiently catalyzed by an evolutionary related enzyme. Alternative activities could have played an important role in the diversification of enzymes by providing a duplicated gene a head start towards being captured by adaptive evolution.
Protein arginine methylation has been implicated in signal transduction, nuclear transport and transcription regulation. Protein arginine methyltransferases (PRMTs) mediate the AdoMet-dependent methylation of many proteins, including many RNA binding proteins involved in various aspects of RNA processing and/or transport. Here we describe the crystal structure of the rat PRMT3 catalytic core in complex with reaction product AdoHcy, determined at 2.0 A resolution. The results reveal a two-domain structure: an AdoMet-binding domain and a barrel-like domain. The AdoMet-binding domain is a compact version of the consensus AdoMet-dependent methyltransferase fold. The active site is situated in a cone-shaped pocket between the two domains. The residues that make up the active site are conserved across the PRMT family, consisting of a double-E loop containing two invariant Glu and one His-Asp proton-relay system. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the PRMT family. In addition, crystal packing and solution behavior suggest dimer formation of the PRMT3 core.
A family of RNA m(5)C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m(5)C 967 MTase, Fmu, as an initial probe. The RNA m(5)C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m(5)C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m(5)U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m(5)C MTase uses a different Cys as a catalytic nucleophile than the DNA m(5)C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m(5)C MTases remains unknown.
Formation of isoaspartyl residues is one of several processes that damage proteins as they age. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) is a conserved and nearly ubiquitous enzyme that catalyzes the repair of proteins damaged by isoaspartyl formation.
We have determined the first structure of a PIMT from crystals of the T. maritima enzyme complexed to S-adenosyl-L-homocysteine (AdoHcy) and refined it to 1.8 A resolution. Although PIMT forms one structural unit, the protein can be divided functionally into three subdomains. The central subdomain closely resembles other S-adenosyl-L-methionine-dependent methyltransferases but bears a striking alteration of topological connectivity, which is not shared by any other member of this family. Rather than arranged as a mixed beta sheet with topology 6 upward arrow7 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow, the central sheet of PIMT is reorganized to 7 upward arrow6 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow. AdoHcy is largely buried between the N-terminal and central subdomains by a conserved and largely hydrophobic loop on one rim of the binding cleft, and a conserved Ser/Thr-rich beta strand on the other. The Ser/Thr-rich strand may provide hydrogen bonds for specific interactions with isoaspartyl substrates. The side chain of Ile-206, a conserved residue, crosses the cleft, restricting access to the donor methyl group to a deep well, the putative isoaspartyl methyl acceptor site.
The structure of PIMT reveals a unique modification of the methyltransferase fold along with a site for specific recognition of isoaspartyl substrates. The sequence conservation among PIMTs suggests that the current structure should prove a reliable model for understanding the repair of isoaspartyl damage in all organisms.
The protein sequence and structure databases are now sufficiently representative that strategies nature uses to evolve new catalytic functions can be identified. Groups of divergently related enzymes whose members catalyze different reactions but share a common partial reaction, intermediate, or transition state (mechanistically diverse superfamilies) have been discovered, including the enolase, amidohydrolase, thiyl radical, crotonase, vicinal-oxygen-chelate, and Fe-dependent oxidase superfamilies. Other groups of divergently related enzymes whose members catalyze different overall reactions that do not share a common mechanistic strategy (functionally distinct suprafamilies) have also been identified: (a) functionally distinct suprafamilies whose members catalyze successive transformations in the tryptophan and histidine biosynthetic pathways and (b) functionally distinct suprafamilies whose members catalyze different reactions in different metabolic pathways. An understanding of the structural bases for the catalytic diversity observed in super- and suprafamilies may provide the basis for discovering the functions of proteins and enzymes in new genomes as well as provide guidance for in vitro evolution/engineering of new enzymes.
Twenty AdoMet-dependent methyltransferases (MTases) have been characterized structurally by X-ray crystallography and NMR. These include seven DNA MTases, five RNA MTases, four protein MTases and four small molecule MTases acting on the carbon, oxygen or nitrogen atoms of their substrates. The MTases share a common core structure of a mixed seven-stranded beta-sheet (6 downward arrow 7 upward arrow 5 downward arrow 4 downward arrow 1 downward arrow 2 downward arrow 3 downward arrow) referred to as an 'AdoMet-dependent MTase fold', with the exception of a protein arginine MTase which contains a compact consensus fold lacking the antiparallel hairpin strands (6 downward arrow 7 upward arrow). The consensus fold is useful to identify hypothetical MTases during structural proteomics efforts on unannotated proteins. The same core structure works for very different classes of MTase including those that act on substrates differing in size from small molecules (catechol or glycine) to macromolecules (DNA, RNA and protein). DNA MTases use a 'base flipping' mechanism to deliver a specific base within a DNA molecule into a typically concave catalytic pocket. Base flipping involves rotation of backbone bonds in double-stranded DNA to expose an out-of-stack nucleotide, which can then be a substrate for an enzyme-catalyzed chemical reaction. The phenomenon is fully established for DNA MTases and for DNA base excision repair enzymes, and is likely to prove general for enzymes that require access to unpaired, mismatched or damaged nucleotides within base-paired regions in DNA and RNA. Several newly discovered MTase families in eukaryotes (DNA 5mC MTases and protein arginine and lysine MTases) offer new challenges in the MTase field.
Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT.
We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 A. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent.
An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.
A systematic computational analysis of protein sequences containing known nuclear domains led to the identification of 28 novel domain families. This represents a 26% increase in the starting set of 107 known nuclear domain families used for the analysis. Most of the novel domains are present in all major eukaryotic lineages, but 3 are species specific. For about 500 of the 1200 proteins that contain these new domains, nuclear localization could be inferred, and for 700, additional features could be predicted. For example, we identified a new domain, likely to have a role downstream of the unfolded protein response; a nematode-specific signalling domain; and a widespread domain, likely to be a noncatalytic homolog of ubiquitin-conjugating enzymes.
A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The Km for iodide and SAM were 12 mM and 12 microM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I-, Br-, and Cl- to corresponding monohalomethanes and of bisulfide to methyl mercaptan.
Ab initio and density functional calculations have been carried out to more fully understand the factors controlling the catalytic activity of the Thermus aquaticus DNA methyltransferase (MTaqI) in the N-methylation at the N(6) of an adenine nucleobase. The noncatalyzed reaction was modeled as a methyl transfer from trimethylsulfonium to the N(6) of adenine. Activation barriers of 32.0 kcal/mol and 24.0 kcal/mol were predicted for the noncatalyzed reaction in the gas phase by MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. Calculations performed to evaluate the effect of substrate positioning in the active site of MTaqI on the reaction determine the barrier to be 23.4 kcal/mol and 17.3 kcal/mol for the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) gas phase calculations, respectively. The effect of hydrogen bonding between the N(6) of adenine and the terminal oxygen of Asn-105 on the activation barrier was also studied. A formamide molecule was modeled into the system to mimic the function of active site residue Asn-105. The activation barrier for this reaction was found to be 21.8 kcal/mol and 15.8 kcal/mol as determined from the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. This result predicts a contribution of less than 2 kcal/mol to the lowering of the activation barrier from amide hydrogen bonding between formamide and N(6) of adenine. Comparison of the reaction coordinates suggest that it is not the hydrogen bonding of the Asn-105 that lends to the catalytic prowess of the enzyme since the organization of the substrates in the active site of the enzyme has a far greater effect on reducing the activation barrier. The results also suggest a stepwise mechanism for the removal of the hydrogen from the N(6) of adenine as opposed to a concerted reaction in which a proton is abstracted simultaneously with the transfer of the methyl group. The hydrogen on the N(6) of the intermediate methyl adenine product is far more acidic than in the reactant complex and may be subsequently abstracted by basic groups in the active site that are too weak to abstract the proton before the full sp(3) hybridization of the attacking nitrogen.
Kinases are a ubiquitous group of enzymes that catalyze the phosphoryl transfer reaction from a phosphate donor (usually ATP) to a receptor substrate. Although all kinases catalyze essentially the same phosphoryl transfer reaction, they display remarkable diversity in their substrate specificity, structure, and the pathways in which they participate. In order to learn the relationship between structural fold and functional specificities in kinases, we have done a comprehensive survey of all available kinase sequences (>17,000) and classified them into 30 distinct families based on sequence similarities. Of these families, 19, covering nearly 98% of all sequences, fall into seven general structural folds for which three-dimensional structures are known. These fold groups include some of the most widespread protein folds, such as Rossmann fold, ferredoxin fold, ribonuclease H fold, and TIM beta/alpha-barrel. On the basis of this classification system, we examined the shared substrate binding and catalytic mechanisms as well as variations of these mechanisms in the same fold groups. Cases of convergent evolution of identical kinase activities occurring in different folds are discussed.
AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltranferase (HKMT), determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.
Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.
Protein lysine methylation by SET domain enzymes regulates chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. The 2.6 A resolution structure of Rubisco large subunit methyltransferase in a pseudo-bisubstrate complex with S-adenosylhomocysteine and a HEPES ion reveals an all-beta architecture for the SET domain embedded within a larger alpha-helical enzyme fold. Conserved regions of the SET domain bind S-adenosylmethionine and substrate lysine at two sites connected by a pore. We propose that methyl transfer is catalyzed by a conserved Tyr at a narrow pore connecting the sites. The cofactor enters by a "back door" on the opposite side of the enzyme from substrate, promoting highly specific protein recognition and allowing addition of multiple methyl groups.
Proteins bearing the widely distributed SET domain have been shown to methylate lysine residues in histones and other proteins. In this issue, three-dimensional structures are reported for three very different SET domain-containing proteins. The structures reveal novel folds for several new domains, including SET, and provide early insights into mechanisms of catalysis and molecular recognition in this family of enzymes.
In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2'O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.