AdoMet-dependent Methyltransferases Research Articles

Profiling the compendium of changes in Saccharomyces cerevisiae due to mutations that alter availability of the main methyl donor S-Adenosylmethionine.

The SAM1 and SAM2 genes encode for S-Adenosylmethionine (AdoMet) synthetase enzymes, with AdoMet serving as the main cellular methyl donor. We have previously shown that independent deletion of these genes alters chromosome stability and AdoMet concentrations in opposite ways in Saccharomyces cerevisiae. To characterize other changes occurring in these mutants, we grew wildtype, sam1Δ/sam1Δ, and sam2Δ/sam2Δ strains in 15 different Phenotypic Microarray plates with different components and measured growth variations. RNA-Sequencing was also carried out on these strains and differential gene expression determined for each mutant. We explored how the phenotypic growth differences are linked to the altered gene expression, and hypothesize mechanisms by which loss of the SAM genes and subsequent AdoMet level changes, impact pathways and processes. We present 6 stories, discussing changes in sensitivity or resistance to azoles, cisplatin, oxidative stress, arginine biosynthesis perturbations, DNA synthesis inhibitors, and tamoxifen, to demonstrate the power of this novel methodology to broadly profile changes due to gene mutations. The large number of conditions that result in altered growth, as well as the large number of differentially expressed genes with wide-ranging functionality, speaks to the broad array of impacts that altering methyl donor abundance can impart. Our findings demonstrate that some cellular changes are directly related to AdoMet-dependent methyltransferases and AdoMet availability, some are directly linked to the methyl cycle and its role in production of several important cellular components, and others reveal impacts of SAM gene mutations on previously unconnected pathways.

Chemical Expansion of the Methyltransferase Reaction: Tools for DNA Labeling and Epigenome Analysis.

DNA is the genetic matter of life composed of four major nucleotides which can be further furnished with biologically important covalent modifications. Among the variety of enzymes involved in DNA metabolism, AdoMet-dependent methyltransferases (MTases) combine the recognition of specific sequences and covalent methylation of a target nucleotide. The naturally transferred methyl groups play important roles in biological signaling, but they are poor physical reporters and largely resistant to chemical derivatization. Therefore, an obvious strategy to unlock the practical utility of the methyltransferase reactions is to enable the transfer of "prederivatized" (extended) versions of the methyl group.However, previous enzymatic studies of extended AdoMet analogs indicated that the transalkylation reactions are drastically impaired as the size of the carbon chain increases. In collaborative efforts, we proposed that, akin to enhanced SN2 reactivity of allylic and propargylic systems, addition of a π orbital next to the transferable carbon atom might confer the needed activation of the reaction. Indeed, we found that MTase-catalyzed transalkylations of DNA with cofactors containing a double or a triple C-C bond in the β position occurred in a robust and sequence-specific manner. Altogether, this breakthrough approach named mTAG (methyltransferase-directed transfer of activated groups) has proven instrumental for targeted labeling of DNA and other types of biomolecules (using appropriate MTases) including RNA and proteins.Our further work focused on the propargylic cofactors and their reactions with DNA cytosine-5 MTases, a class of MTases common for both prokaryotes and eukaryotes. Here, we learned that the 4-X-but-2-yn-1-yl (X = polar group) cofactors suffered from a rapid loss of activity in aqueous buffers due to susceptibility of the triple bond to hydration. This problem was remedied by synthetically increasing the separation between X and the triple bond from one to three carbon units (6-X-hex-2-ynyl cofactors). To further optimize the transfer of the bulkier groups, we performed structure-guided engineering of the MTase cofactor pocket. Alanine replacements of two conserved residues conferred substantial improvements of the transalkylation activity with M.HhaI and three other engineered bacterial C5-MTases. Of particular interest were CpG-specific DNA MTases (M.SssI), which proved valuable tools for studies of mammalian methylomes and chemical probing of DNA function.Inspired by the successful repurposing of bacterial enzymes, we turned to more complex mammalian C5-MTases (Dnmt1, Dnmt3A, and Dnmt3B) and asked if they could ultimately lead to mTAG labeling inside mammalian cells. Our efforts to engineer mouse Dnmt1 produced a variant (Dnmt1*) that enabled efficient Dnmt1-directed deposition of 6-azide-hexynyl groups on DNA in vitro. CRISPR-Cas9 editing of the corresponding codons in the genomic Dnmt1 alleles established endogenous expression of Dnmt1* in mouse embryonic stem cells. To circumvent the poor cellular uptake of AdoMet and its analogs, we elaborated their efficient internalization by electroporation, which has finally enabled selective catalysis-dependent azide tagging of natural Dnmt1 targets in live mammalian cells. The deposited chemical groups were then exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. These findings offer unprecedented inroads into studies of DNA methylation in a wide range of eukaryotic model systems.

Synthesis and Activity of Triazole-Adenosine Analogs as Protein Arginine Methyltransferase 5 Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is an attractive molecular target in anticancer drug discovery due to its extensive involvement in transcriptional control, RNA processing, and other cellular pathways that are causally related to tumor initiation and progression. In recent years, various compounds have been screened or designed to target either the substrate- or cofactor-binding site of PRMT5. To expand the diversity of chemotypes for inhibitory binding to PRMT5 and other AdoMet-dependent methyltransferases, in this work, we designed a series of triazole-containing adenosine analogs aimed at targeting the cofactor-binding site of PRMT5. Triazole rings have commonly been utilized in drug discovery due to their ease of synthesis and functionalization as bioisosteres of amide bonds. Herein, we utilized the electronic properties of the triazole ring as a novel way to specifically target the cofactor-binding site of PRMT5. A total of about 30 compounds were synthesized using the modular alkyne-azide cycloaddition reaction. Biochemical tests showed that these compounds exhibited inhibitory activity of PRMT5 at varying degrees and several showed single micromolar potency, with clear selectivity for PRMT5 over PRMT1. Docking-based structural analysis showed that the triazole ring plays a key role in binding to the characteristic residue Phe327 in the active pocket of PRMT5, explaining the compounds’ selectivity for this type-II enzyme. Overall, this work provides new structure–activity relationship information on the design of AdoMet analogs for selective inhibition of PRMT5. Further structural optimization work will further improve the potency of the top leads.

Selective chemical tracking of Dnmt1 catalytic activity in live cells

SummaryEnzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as “click” handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems.

Human METTL7B is an alkyl thiol methyltransferase that metabolizes hydrogen sulfide and captopril

Methylation of alkyl thiols is a biotransformation pathway designed to reduce thiol reactivity and potential toxicity, yet the gene and protein responsible for human alkyl thiol methyltransferase (TMT) activity remain unknown. Here we demonstrate with a range of experimental approaches using cell lines, in vitro systems, and recombinantly expressed enzyme, that human methyltransferase-like protein 7B (METTL7B) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to hydrogen sulfide (H2S) and other exogenous thiol small molecules. METTL7B gene modulation experiments, including knockdown in HepG2 cells and overexpression in HeLa cells, directly alter the methylation of the drug captopril, a historic probe substrate for TMT activity. Furthermore, recombinantly expressed and purified wild-type METTL7B methylates several thiol compounds, including H2S, 7α-thiospironolactone, l-penicillamine, and captopril, in a time- and concentration-dependent manner. Typical for AdoMet-dependent small molecule methyltransferases, S-adenosyl-l-homocysteine (AdoHcy) inhibited METTL7B activity in a competitive fashion. Similarly, mutating a conserved aspartate residue, proposed to anchor AdoMet into the active site, to an alanine (D98A) abolished methylation activity. Endogenous thiols such as glutathione and cysteine, or classic substrates for other known small molecule S-, N-, and O-methyltransferases, were not substrates for METTL7B. Our results confirm, for the first time, that METTL7B, a gene implicated in multiple disease states including rheumatoid arthritis and breast cancer, encodes a protein that methylates small molecule alkyl thiols. Identifying the catalytic function of METTL7B will enable future pharmacological research in disease pathophysiology where altered METTL7B expression and, potentially H2S levels, can disrupt cell growth and redox state.

Crystallographic and Computational Characterization of Methyl Tetrel Bonding in S-Adenosylmethionine-Dependent Methyltransferases.

Tetrel bonds represent a category of non-bonding interaction wherein an electronegative atom donates a lone pair of electrons into the sigma antibonding orbital of an atom in the carbon group of the periodic table. Prior computational studies have implicated tetrel bonding in the stabilization of a preliminary state that precedes the transition state in SN2 reactions, including methyl transfer. Notably, the angles between the tetrel bond donor and acceptor atoms coincide with the prerequisite geometry for the SN2 reaction. Prompted by these findings, we surveyed crystal structures of methyltransferases in the Protein Data Bank and discovered multiple instances of carbon tetrel bonding between the methyl group of the substrate S-adenosylmethionine (AdoMet) and electronegative atoms of small molecule inhibitors, ions, and solvent molecules. The majority of these interactions involve oxygen atoms as the Lewis base, with the exception of one structure in which a chlorine atom of an inhibitor functions as the electron donor. Quantum mechanical analyses of a representative subset of the methyltransferase structures from the survey revealed that the calculated interaction energies and spectral properties are consistent with the values for bona fide carbon tetrel bonds. The discovery of methyl tetrel bonding offers new insights into the mechanism underlying the SN2 reaction catalyzed by AdoMet-dependent methyltransferases. These findings highlight the potential of exploiting these interactions in developing new methyltransferase inhibitors.

TrmD: A Methyl Transferase for tRNA Methylation With m1G37.

TrmD is an S-adenosyl methionine (AdoMet)-dependent methyl transferase that synthesizes the methylated m1G37 in tRNA. TrmD is specific to and essential for bacterial growth, and it is fundamentally distinct from its eukaryotic and archaeal counterpart Trm5. TrmD is unusual by using a topological protein knot to bind AdoMet. Despite its restricted mobility, the TrmD knot has complex dynamics necessary to transmit the signal of AdoMet binding to promote tRNA binding and methyl transfer. Mutations in the TrmD knot block this intramolecular signaling and decrease the synthesis of m1G37-tRNA, prompting ribosomes to +1-frameshifts and premature termination of protein synthesis. TrmD is unique among AdoMet-dependent methyl transferases in that it requires Mg2+ in the catalytic mechanism. This Mg2+ dependence is important for regulating Mg2+ transport to Salmonella for survival of the pathogen in the host cell. The strict conservation of TrmD among bacterial species suggests that a better characterization of its enzymology and biology will have a broad impact on our understanding of bacterial pathogenesis.

Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases.

Identifying an enzyme's substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.

Identifying novel selective non-nucleoside DNA methyltransferase 1 inhibitors through docking-based virtual screening.

The DNA methyltransferases (DNMTs) found in mammals include DNMT1, DNMT3A, and DNMT3B and are attractive targets in cancer chemotherapy. DNMT1 was the first among the DNMTs to be characterized, and it is responsible for maintaining DNA methylation patterns. A number of DNMT inhibitors have been reported, but most of them are nucleoside analogs that can lead to toxic side effects and lack specificity. By combining docking-based virtual screening with biochemical analyses, we identified a novel compound, DC_05. DC_05 is a non-nucleoside DNMT1 inhibitor with low micromolar IC50 values and significant selectivity toward other AdoMet-dependent protein methyltransferases. Through a process of similarity-based analog searching, compounds DC_501 and DC_517 were found to be more potent than DC_05. These three potent compounds significantly inhibited cancer cell proliferation. The structure-activity relationship (SAR) and binding modes of these inhibitors were also analyzed to assist in the future development of more potent and more specific DNMT1 inhibitors.

Purification, crystallization and preliminary crystallographic analysis of the 16S rRNA methyltransferase RsmI from Escherichia coli.

RsmI and RsmH are AdoMet-dependent methyltransferases that are responsible for the 2'-O-methylation and N(4)-methylation of C1402 of Escherichia coli 16S rRNA, respectively. Modification of this site has been found to play a role in fine-tuning the shape and function of the P-site to increase the decoding fidelity. It is interesting to study the mechanism by which C1402 can be methylated by both RsmI and RsmH. The crystal structure of RsmH in complex with AdoMet and cytidine has recently been determined and provided some implications for N(4)-methylation of this site. Here, the purification and crystallization of RsmI as well as its preliminary crystallographic analysis are reported. Co-crystallization of RsmI with AdoMet was carried out by the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 2.60 Å resolution on beamline 1W2B at BSRF. The crystal contained three molecules per asymmetric unit and belonged to space group C2, with unit-cell parameters a = 121.9, b = 152.5, c = 54.2 Å, β = 93.4°.

Evolutionary and sequence-based relationships in bacterial AdoMet-dependent non-coding RNA methyltransferases.

BackgroundRNA post-transcriptional modification is an exciting field of research that has evidenced this editing process as a sophisticated epigenetic mechanism to fine tune the ribosome function and to control gene expression. Although tRNA modifications seem to be more relevant for the ribosome function and cell physiology as a whole, some rRNA modifications have also been seen to play pivotal roles, essentially those located in central ribosome regions. RNA methylation at nucleobases and ribose moieties of nucleotides appear to frequently modulate its chemistry and structure. RNA methyltransferases comprise a superfamily of highly specialized enzymes that accomplish a wide variety of modifications. These enzymes exhibit a poor degree of sequence similarity in spite of using a common reaction cofactor and modifying the same substrate type.ResultsRelationships and lineages of RNA methyltransferases have been extensively discussed, but no consensus has been reached. To shed light on this topic, we performed amino acid and codon-based sequence analyses to determine phylogenetic relationships and molecular evolution. We found that most Class I RNA MTases are evolutionarily related to protein and cofactor/vitamin biosynthesis methyltransferases. Additionally, we found that at least nine lineages explain the diversity of RNA MTases. We evidenced that RNA methyltransferases have high content of polar and positively charged amino acid, which coincides with the electrochemistry of their substrates.ConclusionsAfter studying almost 12,000 bacterial genomes and 2,000 patho-pangenomes, we revealed that molecular evolution of Class I methyltransferases matches the different rates of synonymous and non-synonymous substitutions along the coding region. Consequently, evolution on Class I methyltransferases selects against amino acid changes affecting the structure conformation.

Selective Non-nucleoside Inhibitors of Human DNA Methyltransferases Active in Cancer Including in Cancer Stem Cells

DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.

Crystal structure of the N‐terminal methyltransferase‐like domain of anamorsin

Anamorsin is a recently identified molecule that inhibits apoptosis during hematopoiesis. It contains an N-terminal methyltransferase-like domain and a C-terminal Fe-S cluster motif. Not much is known about the function of the protein. To better understand the function of anamorsin, we have solved the crystal structure of the N-terminal domain at 1.8 Å resolution. Although the overall structure resembles a typical S-adenosylmethionine (SAM) dependent methyltransferase fold, it lacks one α-helix and one β-strand. As a result, the N-terminal domain as well as the full-length anamorsin did not show S-adenosyl-L-methionine (AdoMet) dependent methyltransferase activity. Structural comparisons with known AdoMet dependent methyltransferases reveals subtle differences in the SAM binding pocket that preclude the N-terminal domain from binding to AdoMet. The N-terminal methyltransferase-like domain of anamorsin probably functions as a structural scaffold to inhibit methyl transfers by out-competing other AdoMet dependant methyltransferases or acts as bait for protein-protein interactions.

A Novel Small Molecule Methyltransferase Is Important for Virulence in Candida albicans

Candida albicans is an opportunistic pathogen capable of causing life-threatening infections in immunocompromised individuals. Despite its significant health impact, our understanding of C. albicans pathogenicity is limited, particularly at the molecular level. One of the largely understudied enzyme families in C. albicans are small molecule AdoMet-dependent methyltransferases (smMTases), which are important for maintenance of cellular homeostasis by clearing toxic chemicals, generating novel cellular intermediates, and regulating intra- and interspecies interactions. In this study, we demonstrated that C. albicans Crg1 (CaCrg1) is a bona fide smMTase that interacts with the toxin in vitro and in vivo. We report that CaCrg1 is important for virulence-related processes such as adhesion, hyphal elongation, and membrane trafficking. Biochemical and genetic analyses showed that CaCrg1 plays a role in the complex sphingolipid pathway: it binds to exogenous short-chain ceramides in vitro and interacts genetically with genes of glucosylceramide pathway, and the deletion of CaCRG1 leads to significant changes in the abundance of phytoceramides. Finally we found that this novel lipid-related smMTase is required for virulence in the waxmoth Galleria mellonella, a model of infection.

Conservation and Functional Importance of Carbon–Oxygen Hydrogen Bonding in AdoMet-Dependent Methyltransferases

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

The Temperature Sensitivity of a Mutation in the Essential tRNA Modification Enzyme tRNA Methyltransferase D (TrmD)

Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m(1)G37 on the 3' side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.

Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure

Archaeal and eukaryotic tRNA (N(2),N(2)-guanine)-dimethyltransferase (Trm1) produces N(2),N(2)-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.

Investigation of the Molecular Origins of Protein-arginine Methyltransferase I (PRMT1) Product Specificity Reveals a Role for Two Conserved Methionine Residues

Protein-arginine methyltransferases aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is due in part to the diverse product specificity displayed by the protein-arginine methyltransferases. In addition to site location within a protein, biological response is also determined by the degree (mono-/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here, we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity but also control substrate specificity. Mutation of Met-155 or Met-48 results in a loss in activity and a change in distribution of mono- and dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is much slower than transfer of the first methyl group in M48L, especially for arginine residues located in the center of the peptide substrate where turnover of the monomethylated species is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.

Hypermethioninemias of genetic and non‐genetic origin: A review

This review covers briefly the major conditions, genetic and non-genetic, sometimes leading to abnormally elevated methionine, with emphasis on recent developments. A major aim is to assist in the differential diagnosis of hypermethioninemia. The genetic conditions are: (1) Homocystinuria due to cystathionine β-synthase (CBS) deficiency. At least 150 different mutations in the CBS gene have been identified since this deficiency was established in 1964. Hypermethioninemia is due chiefly to remethylation of the accumulated homocysteine. (2) Deficient activity of methionine adenosyltransferases I and III (MAT I/III), the isoenzymes the catalytic subunit of which are encoded by MAT1A. Methionine accumulates because its conversion to S-adenosylmethionine (AdoMet) is impaired. (3) Glycine N-methyltrasferase (GNMT) deficiency. Disruption of a quantitatively major pathway for AdoMet disposal leads to AdoMet accumulation with secondary down-regulation of methionine flux into AdoMet. (4) S-adenosylhomocysteine (AdoHcy) hydrolase (AHCY) deficiency. Not being catabolized normally, AdoHcy accumulates and inhibits many AdoMet-dependent methyltransferases, producing accumulation of AdoMet and, thereby, hypermethioninemia. (5) Citrin deficiency, found chiefly in Asian countries. Lack of this mitochondrial aspartate-glutamate transporter may produce (usually transient) hypermethioninemia, the immediate cause of which remains uncertain. (6) Fumarylacetoacetate hydrolase (FAH) deficiency (tyrosinemia type I) may lead to hypermethioninemia secondary either to liver damage and/or to accumulation of fumarylacetoacetate, an inhibitor of the high K(m) MAT. Additional possible genetic causes of hypermethioninemia accompanied by elevations of plasma AdoMet include mitochondrial disorders (the specificity and frequency of which remain to be elucidated). Non-genetic conditions include: (a) Liver disease, which may cause hypermethioninemia, mild, or severe. (b) Low-birth-weight and/or prematurity which may cause transient hypermethioninemia. (c) Ingestion of relatively large amounts of methionine which, even in full-term, normal-birth-weight babies may cause hypermethioninemia.

Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion toS-Adenosyl-L-Homocysteine the Key to Deregulated Lipid Metabolism?

Homocysteine (Hcy) has been recognized for the past five decades as a risk factor for atherosclerosis. However, the role of Hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by Hcy accumulation is poorly understood. Due to the reversal of the physiological direction of the reaction catalyzed by S-adenosyl-L-homocysteine hydrolase Hcy accumulation leads to the synthesis of S-adenosyl-L-homocysteine (AdoHcy). AdoHcy is a strong product inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and to date more than 50 AdoMet-dependent methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids have been identified. Phospholipid methylation is the major consumer of AdoMet, both in mammals and in yeast. AdoHcy accumulation induced either by Hcy supplementation or due to S-adenosyl-L-homocysteine hydrolase deficiency results in inhibition of phospholipid methylation in yeast. Moreover, yeast cells accumulating AdoHcy also massively accumulate triacylglycerols (TAG). Similarly, Hcy supplementation was shown to lead to increased TAG and sterol synthesis as well as to the induction of the unfolded protein response (UPR) in mammalian cells. In this review a model of deregulation of lipid metabolism in response to accumulation of AdoHcy in Hcy-associated pathology is proposed.