Methyl Transfer Research Articles

Genome-wide lone strand adenine methylation in Deinococcus radiodurans R1: Regulation of gene expression through DR0643-dependent adenine methylation.

DNA methylation is a covalent modification of adenine or cytosine in the genome of an organism and is found in diverse microbes including the radiation resistant bacterium Deinococcus radiodurans R1. Although earlier findings have confirmed repression or de-repression of certain genes in adenine methyltransferase (DR_0643/Dam1DR) deficient D. radiodurans mutant however, the overall regulatory aspects of Dam1DR-mediated adenine methylation remain mostly unexplored. In the present study, we compared the genome-wide methylome and the corresponding transcriptome of D. radiodurans WT and Δdam1 mutant to explore the correlation between methylation and gene expression. In D. radiodurans, deletion of DR_0643 ORF (Δdam1) led to hypomethylation of 512 genes resulting in differential expression of 168 genes (99 genes are upregulated and 69 genes are downregulated). The modification patterns deduced for Dam1DR (DR_0643) and Dam2DR (DR_2267) were non-palindromic and atypical. Moreover, we observed methylation at opportunistic sites that show adenine methylation only in D. radiodurans Δdam1 and not in D. radiodurans WT. Correlation between the methylome and transcriptome suggests that hypomethylation at Dam1DR specific sites had both negative as well as a positive effects on gene expression. Pathways such as amino acid metabolism, transport, oxidative phosphorylation, quorum sensing, signal transduction, two-component system, glycolysis/gluconeogenesis, TCA cycle, glyoxylate and dicarboxylate metabolism were modulated by Dam1DR-mediated adenine methylation in D. radiodurans. Processes such as DNA repair, recombination, ATPase and transmembrane transporter activity were enriched when Dam1DR mutant was subjected to radiation stress. We further evaluated the molecular interactions and mode of binding between Dam1DR protein and S-adenosyl methionine using molecular docking followed by MD simulation. To get a better insight into the methylation mechanism, the Dam1DR-SAM complex was also docked with a DNA molecule to elucidate DNA-Dam1DR structural interaction during methyl-group transfer reaction. In summary, our work presents comprehensive and integrative approaches to investigate both functional and structural aspects of DNA adenine methyltransferase (Dam1DR) in D. radiodurans biology.

MdMTA-mediated m6 A modification enhances drought tolerance by promoting mRNA stability and translation efficiency of genes involved in lignin deposition and oxidative stress.

Although the N6 -methyladenosine (m6 A) modification is the most prevalent RNA modification in eukaryotes, the global m6 A modification landscape and its molecular regulatory mechanism in response to drought stress remain unclear. Transcriptome-wide m6 A methylome profiling revealed that m6 A is mainly enriched in the coding sequence and 3' untranslated region in response to drought stress in apple, by recognizing the plant-specific sequence motif UGUAH (H=A, U or C). We identified a catalytically active component of the m6 A methyltransferase complex, MdMTA. An in vitro methyl transfer assay, dot blot, LC-MS/MS and m6 A-sequencing (m6 A-seq) suggested that MdMTA is an m6 A writer and essential for m6 A mRNA modification. Further studies revealed that MdMTA is required for apple drought tolerance. m6 A-seq and RNA-seq analyses under drought conditions showed that MdMTA mediates m6 A modification and transcripts of mRNAs involved in oxidative stress and lignin deposition. Moreover, m6 A modification promotes mRNA stability and the translation efficiency of these genes in response to drought stress. Consistently, MdMTA enhances lignin deposition and scavenging of reactive oxygen species under drought conditions. Our results reveal the global involvement of m6 A modification in the drought response of perennial apple trees and illustrate its molecular mechanisms, thereby providing candidate genes for the breeding of stress-tolerant apple cultivars.

Unraveling the Origins of Changing Product Specificity Properties of Arginine Methyltransferase PRMT7 by the E181D and E181D/Q329A Mutations through QM/MM MD and Free-Energy Simulations.

Arginine methylations can regulate important biological processes and affect many cellular activities, and the enzymes that catalyze the methylations are protein arginine methyltransferases (PRMTs). The biological consequences of arginine methylations depend on the methylation states of arginine that are determined by the PRMT's product specificity. Nevertheless, it is still unclear how different PRMTs may generate different methylation states for the target proteins. PRMT7 is the only known member of type III PRMT that produces monomethyl arginine (MMA) product. Interestingly, its E181D and E181D/Q329A mutants can catalyze, respectively, the formation of asymmetrically dimethylated arginine (ADMA) and symmetrically dimethylated arginine (SDMA). The reasons as to why the mutants have the abilities to add the second methyl group and E181D (E181D/Q329A) has the unique product specificity in generating ADMA (SDMA) have not been understood. Here, quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and potential of mean force (PMF) free-energy simulations are performed for the E181D and E181D/Q329A mutants to understand the origin for their ability to generate, respectively, ADMA and SDMA. The simulations show that the free-energy barrier for adding the second methyl group to MMA in E181D (E181D/Q329A) to produce ADMA (SDMA) is considerably lower than the corresponding barriers in wild type and E181D/Q329A (wild type and E181D), consistent with experimental observations. Some important factors that contribute to the change of the activity and product specificity due to the E181D and E181D/Q329A mutations are identified based on the data from the simulations and analysis. It is shown that the transferable methyl group (from SAM) and Nη2 (the nitrogen atom that is methylated in the substrate MMA) can only form good near-attack conformations in the E181D reaction state for the methyl transfer (not in wild type and E181D/Q329A), while the transferable methyl group and Nη1 (the nitrogen atom that is not methylated in the substrate MMA) can only form good near-attack conformations in E181D/Q329A (not in wild type and E181D). The results suggest that the steric repulsions in the reaction state between the methyl group on MMA and active-site residues (e.g., Q329) and the release of such repulsions (e.g., from the Q329A mutation) may play an important role in generating specific near-attack conformations for the methyl transfer and controlling the product specificity for the mutants. The general principle identified in this work for PRMT7 is expected to be useful for understanding the activity and product specificity of other PRMTs as well.

Structure and mechanism of the methyltransferase ribozyme MTR1.

RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m1A) in RNA, using O6-methylguanine (m6G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.

Structure and mechanism of a methyltransferase ribozyme

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

Functional Study of Leishmania braziliensis Protein Arginine Methyltransferases (PRMTs) Reveals That PRMT1 and PRMT5 Are Required for Macrophage Infection.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other RBP interactions by transferring methyl groups to arginine residues (R-methylation). Herein, we functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that R-methylation profiles vary among Leishmania species and across L.braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes. A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation assay using HA-tagged PRMTs, suggesting a network of putative targets of PRMTs and cooperation between the R-methylation writers. Knockout of each L.braziliensis PRMT led to significant changes in global arginine methylation patterns without affecting cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine levels. Finally, we demonstrate that L.braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and for axenic amastigote proliferation. The results indicate that R-methylation is modulated across lifecycle stages in L.braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L.braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

Factors That Determine the Variation of Equilibrium and Kinetic Properties of QM/MM Enzyme Simulations: QM Region, Conformation, and Boundary Condition.

To analyze the impact of various technical details on the results of quantum mechanical (QM)/molecular mechanical (MM) enzyme simulations, including the QM region size, catechol-O-methyltransferase (COMT) is studied as a model system using an approximate QM/MM method (DFTB3/CHARMM). The results show that key equilibrium and kinetic properties for methyl transfer in COMT exhibit limited variations with respect to the size of the QM region, which ranges from ∼100 to ∼500 atoms in this study. With extensive sampling, local and global structural characteristics of the enzyme are largely conserved across the studied QM regions, while the nature of the transition state (e.g., secondary kinetic isotope effect) and reaction exergonicity are largely maintained. Deviations in the free energy profile with different QM region sizes are similar in magnitude to those observed with changes in other simulation protocols, such as different initial enzyme conformations and boundary conditions. Electronic structural properties, such as the covariance matrix of residual charge fluctuations, appear to exhibit rather long-range correlations, especially when the peptide backbone is included in the QM region; this observation holds when a range-separated DFT approach is used as the QM region, suggesting that delocalization error is unlikely the origin. Overall, the analyses suggest that multiple simulation details determine the results of QM/MM enzyme simulations with comparable contributions.

Solvent Effects on the Menshutkin Reaction.

The Menshutkin reaction is a methyl transfer reaction relevant in fields ranging from biochemistry to chemical synthesis. In the present work, the energetics and solvent distributions for NH3+MeCl and Pyr+MeBr reactions were investigated in explicit solvent (water, methanol, acetonitrile, benzene, cyclohexane) by means of reactive molecular dynamics simulations. For polar solvents (water, methanol, and acetonitrile) and benzene, strong to moderate catalytic effects for both reactions were found, whereas apolar and bulky cyclohexane interacts weakly with the solute and does not show pronounced barrier reduction. The calculated barrier heights for the Pyr+MeBr reaction in acetonitrile and cyclohexane are 23.2 and 28.1 kcal/mol compared with experimentally measured barriers of 22.5 and 27.6 kcal/mol, respectively. The solvent distributions change considerably between reactant and TS but comparatively little between TS and product conformations of the solute. As the system approaches the transition state, correlated solvent motions occur which destabilize the solvent-solvent interactions. This is required for the system to surmount the barrier. Finally, it is found that the average solvent-solvent interaction energies in the reactant, TS, and product state geometries are correlated with changes in the solvent structure around the solute.

DNA methylation: a saga of genome maintenance in hematological perspective

In recent years, an explosion of interest for genome methylation in hematopoietic stem cells (HSCs) differentiation and transplantation has been observed. The differentiation and timely proliferation of HSCs require a stringent regulation of gene expression; this can't be maintained without the regulation of genome methylation. Evidences of changes in genome methylation patterns in healthy and cancer cells have opened a new paradigm for diagnostic and therapeutic strategies. Genome methylation or DNA methylation is achieved by DNA methyltransferases (DNMTs) through the transfer of methyl group leading to the regulation of gene's expression. Till now five DNMTs have been found in mammals, known as DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. In this review, we have carefully evaluated the spacio-temporal landscape expression and role of DNMTs during hematopoiesis, hematopoietic malignancy and HSCs transplantation. Focusing on the connection of DNA methylation with gene expression in association with cellular signalling, histone modifications, it is proposed for further improvement of strategies to use DNA methylation as a marker in clinical diagnosis as well as in therapeutic interventions by applying DNMTs inhibitors.

DNA Labeling Using DNA Methyltransferases.

DNA methyltransferases (MTases) uniquely combine the ability to recognize and covalently modify specific target sequences in DNA using the ubiquitous cofactor S-Adenosyl-L-methionine (AdoMet). Although DNA methylation plays important roles in biological signaling, the transferred methyl group is a poor reporter and is highly inert to further biocompatible derivatization. To unlock the biotechnological power of these enzymes, extended cofactor AdoMet analogs have been developed that enable targeted MTase-directed attachment of larger moieties containing functional or reporter groups onto DNA. As the enlarged cofactors are not always compatible with the active sites of native MTases, steric engineering of the active site has been employed to optimize their alkyltransferase activity. In addition to the described cofactor analogs, recently discovered atypical reactions of DNA cytosine-5 MTases involving non-cofactor-like compounds can also be exploited for targeted derivatization and labeling of DNA. Altogether, these approaches offer new powerful tools for sequence-specific covalent DNA labeling, leading to a variety of useful techniques in DNA research, diagnostics and nanotechnologies, and have already proven practical utility for optical DNA mapping and high-throughput epigenome studies.

A method for the production, purification and liposome reconstitution of cobamide synthase.

Cobamides are essential for the performance of a variety of reactions such methyl transfers, carbon skeleton rearrangements, and eliminations in both prokaryotes and eukaryotes. However, cobamide biosynthesis is limited to a subset of bacteria and archaea. The biosynthesis pathway culminates with the activation and attachment of a lower ligand to the corrin ring; this branch of the pathway is known as nucleotide loop assembly (NLA) pathway. The cobamide synthase (CobS) enzyme is the penultimate step in NLA pathway, and catalyzes the attachment of an α-ribotide to the activated corrin ring. While other NLA enzymes have been well-studied, studies of CobS have proven difficult to date. CobS is an integral membrane protein, and limitations have been largely due to difficulties in protein purification. Here we provide a method to purify CobS, reconstitute protein in proteoliposomes, and assay for its activity.

Changes in ligand coordination mode induce bimetallic C-C coupling pathways.

Carbon-carbon coupling is one of the most powerful tools in the organic synthesis arsenal. Known methodologies primarily exploit monometallic Pd0/PdII catalytic mechanisms to give new C-C bonds. Bimetallic C-C coupling mechanisms that involve a PdI/PdII redox cycle, remain underexplored. Thus, a detailed mechnaistic understanding is imperative for the development of new bimetallic catalysts. Previously, a PdII-Me dimer (1) supported by L1, which has phosphine and 1-azaallyl donor groups, underwent reductive elimination to give ethane, a PdI dimer, a PdII monometallic complex, and Pd black. Herein, a comprehensive experimental and computational study of the reactivity of 1 is presented, which reveals that the versatile coordination chemistry of L1 promotes bimetallic C-C bond formation. The phosphine 1-azaallyl ligand adopts various bridging modes to maintain the bimetallic structure throughout the C-C bond forming mechanism, which involves intramolecular methyl transfer and 1,1-reductive elimination from one of the palladium atoms. The minor byproduct, methane, likely forms through a monometallic intermediate that is sensitive to solvent C-H activation. Overall, the capacity of L1 to adopt different coordination modes promotes the bimetallic C-C coupling channel through pathways that are unattainable with statically-coordinated ligands.

RNA nucleotide methylation: 2021 update.

Among RNA modifications, transfer of methylgroups from the typical cofactor S-adenosyl-l-methionine by methyltransferases (MTases) to RNA is by far the most common reaction. Since our last review about a decade ago, the field has witnessed the re-emergence of mRNA methylation as an important mechanism in gene regulation. Attention has then spread to many other RNA species; all being included into the newly coined concept of the "epitranscriptome." The focus moved from prokaryotes and single cell eukaryotes as model organisms to higher eukaryotes, in particular to mammals. The perception of the field has dramatically changed over the past decade. A previous lack of phenotypes in knockouts in single cell organisms has been replaced by the apparition of MTases in numerous disease models and clinical investigations. Major driving forces of the field include methylation mapping techniques, as well as the characterization of the various MTases, termed "writers." The latter term has spilled over from DNA modification in the neighboring epigenetics field, along with the designations "readers," applied to mediators of biological effects upon specific binding to a methylated RNA. Furthermore "eraser" enzymes effect the newly discovered oxidative removal of methylgroups. A sense of reversibility and dynamics has replaced the older perception of RNA modification as a concrete-cast, irreversible part of RNA maturation. A related concept concerns incompletely methylated residues, which, through permutation of each site, lead to inhomogeneous populations of numerous modivariants. This review recapitulates the major developments of the past decade outlined above, and attempts a prediction of upcoming trends. This article is categorized under: RNA Processing > RNA Editing and Modification.

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction.

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.

One-Pot Synthesis of Polyethylene-Based Block Copolymers via a Dual Polymerization Pathway.

Polyethylene-poly(methyl acrylate) multiblock copolymers were obtained using a catalyst system consisting of a pentamethylcyclopentadienyl cobalt complex (CoIII(η5-C5H5)P(OMe)3I2) and iso-butyl modified methylaluminoxane (MMAO). As the chain-growth mechanism, the self-switching between organometallic-mediated radical polymerization (OMRP) and coordination-insertion polymerization (CIP) is suggested. As a possible polymerization mechanism, we propose that (1) the methyl and iso-butyl groups transfer from Al to Co and the single methyl acrylate (MA) unit insertion into the Me-Co and H-Co allows for the in situ formation of Co-C(COOMe) bonds as an initiator for OMRP of MA, (2) the migratory insertion of ethylene into Co-C(COOMe) bonds leads to the formation of an alkyl-Co species as active species for CIP of ethylene, and (3) MA insertion to the alkyl-Co to regenerate a Co-C(COOMe) bond. The architectures of copolymers were confirmed by various nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA)/differential scanning calorimetry (DSC), and size-exclusion chromatography (SEC) analyses.

Placental transport of parabens studied using an ex-vivo human perfusion model.

Parabens are a group of chemicals widely used as preservatives in daily consumer products such as cosmetics, food items, pharmaceuticals and household commodities. They have been broadly detected in human samples indicating a general human exposure, and concerns arose from their potential endocrine disrupting effect. Especially the exposure to parabens during pregnancy is concerning, as the time of fetal development is a particularly vulnerable period. The aim of this study was to investigate the transport and metabolism of four commonly used parabens: methyl-, ethyl-, propyl- and butylparaben (MeP, EtP, PrP and BuP) and the metabolite para-hydroxybenzoic acid (PHBA) across the human placenta. An ex-vivo human placental perfusion model was used. The test compounds were added in the maternal compartment (with initial concentrations of 1mM or 0.1mM). Placental transport was evaluated by fetal-maternal concentration ratios (FM-ratio), transport index (TI) and indicative permeability (IP). Information about parabens kinetics was taken from 10 perfusions and PHBA from 7 perfusions. Paraben metabolism was not detected. The placental transport of MeP, EtP, PrP, BuP and PHBA revealed a transfer from maternal to fetal circulations with FM120 of 0.86±0.27 (MeP), 0.98±0.28 (EtP), 1.00±0.28 (PrP), 1.12±0.59 (BuP) and 0.82±0.37 (PHBA). The test substances accumulated in the perfused tissue in some degree. The average kinetic parameters FM-ratio, TI and IP were not different between chemicals. The present study shows that the placenta barrier is permeable to all four parabens and the metabolite, which implies potential fetal exposure.

Enhanced Electrocatalytic Detection of Choline Based on CNTs and Metal Oxide Nanomaterials

Choline is an officially established essential nutrient and precursor of the neurotransmitter acetylcholine. It is employed as a cholinergic activity marker in the early diagnosis of brain disorders such as Alzheimer’s and Parkinson’s disease. Low levels of choline in diets and biological fluids, such as blood plasma, urine, cerebrospinal and amniotic fluid, could be an indication of neurological disorder, fatty liver disease, neural tube defects and hemorrhagic kidney necrosis. Meanwhile, it is known that choline metabolism involves oxidation, which frees its methyl groups for entrance into single-C metabolism occurring in three phases: choline oxidase, betaine synthesis and transfer of methyl groups to homocysteine. Electrocatalytic detection of choline is of physiological and pathological significance because choline is involved in the physiological processes in the mammalian central and peripheral nervous systems and thus requires a more reliable assay for its determination in biological, food and pharmaceutical samples. Despite the use of several methods for choline determination, the superior sensitivity, high selectivity and fast analysis response time of bioanalytical-based sensors invariably have a comparative advantage over conventional analytical techniques. This review focuses on the electrocatalytic activity of nanomaterials, specifically carbon nanotubes (CNTs), CNT nanocomposites and metal/metal oxide-modified electrodes, towards choline detection using electrochemical sensors (enzyme and non-enzyme based), and various electrochemical techniques. From the survey, the electrochemical performance of the choline sensors investigated, in terms of sensitivity, selectivity and stability, is ascribed to the presence of these nanomaterials.

245 Greenhouse Gas Emissions Mitigation Strategies

Abstract Livestock production contributed 3.9% to the total greenhouse gas (GHG) emission from the US in 2018. Most studies to mitigate GHG from livestock are focused on enteric methane because it contributes about 70% of all livestock GHG emissions. Mitigation options can be broadly categorized into dietary and rumen manipulation. Enteric methane emissions are strongly correlated to dry matter intake and somewhat sensitive to diet composition. Dietary manipulation methods include increasing feed digestibility, such as concentrate to forage ratio, or increasing fats and oils, which are associated with lower methane emissions. These reduce digestible fiber that are positively related to methane production and more energy passing the rumen without being degraded, respectively. Rumen manipulation through feed additives can be further classified based on the mode of action: 1. rumen environment modifiers indirectly affecting emissions and 2. direct methanogenesis inhibitors. The rumen environment modifiers act on the conditions that promote methanogenesis. These include ionophores, plant bioactive compounds such as essential oils and tannins, and nitrate rich feeds that serve as alternative hydrogen sinks and directly compete with methanogens thereby reducing methane emissions. The inhibitor category include 3-nitroxypropanol and seaweeds containing halogenated compounds. The former was reported to reduce enteric methane emissions (g/d) by 39% in dairy and 22% in beef cattle. Seaweed, in particular Asparagopsis spp., reduced emissions intensity (g/kg milk) by up to 67% in dairy and emissions yield (g/kg dry matter intake) by up to 98% in beef cattle. Because inhibitors are structural analogs of methane, their mode of action is through competitive inhibition of the methyl transfer reaction catalyzed by methyl coenzyme-M reductase, the last enzyme in methanogenesis. The combination of dietary and rumen manipulation options, including feed additives, is expected to reduce enteric methane emissions by over 30% in the next decade without compromising animal productivity and health.

Probing multiple enzymatic methylation events in real time with NMR spectroscopy

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

Repurposing epigenetic inhibitors to target the Clostridioides difficile-specific DNA adenine methyltransferase and sporulation regulator CamA

ABSTRACT Epigenetically targeted therapeutic development, particularly for SAM-dependent methylations of DNA, mRNA and histones has been proceeding rapidly for cancer treatments over the past few years. However, this approach has barely begun to be exploited for developing new antibiotics, despite an overwhelming global need to counter antimicrobial resistance. Here, we explore whether SAM analogues, some of which are in (pre)clinical studies as inhibitors of human epigenetic enzymes, can also inhibit Clostridioides difficile-specific DNA adenine methyltransferase (CamA), a sporulation regulator present in all C. difficile genomes sequenced to date, but found in almost no other bacteria. We found that SGC0946 (an inhibitor of DOT1L), JNJ-64619178 (an inhibitor of PRMT5) and SGC8158 (an inhibitor of PRMT7) inhibit CamA enzymatic activity in vitro at low micromolar concentrations. Structural investigation of the ternary complexes of CamA-DNA in the presence of SGC0946 or SGC8158 revealed conformational rearrangements of the N-terminal arm, with no apparent disturbance of the active site. This N-terminal arm and its modulation of exchanges between SAM (the methyl donor) and SAH (the reaction product) during catalysis of methyl transfer are, to date, unique to CamA. Our work presents a substantial first step in generating potent and selective inhibitors of CamA that would serve in the near term as chemical probes to investigate the cellular mechanism(s) of CamA in controlling spore formation and colonization, and eventually as therapeutic antivirulence agents useful in treating C. difficile infection.