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1.
Nat Commun ; 13(1): 2284, 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35477710

RESUMO

2-iminoacetate synthase ThiH is a radical S-adenosyl-L-methionine (SAM) L-tyrosine lyase and catalyzes the L-tyrosine Cα-Cß bond break to produce dehydroglycine and p-cresol while the radical SAM L-tryptophan lyase NosL cleaves the L-tryptophan Cα-C bond to produce 3-methylindole-2-carboxylic acid. It has been difficult to understand the features that condition one C-C bond break over the other one because the two enzymes display significant primary structure similarities and presumably similar substrate-binding modes. Here, we report the crystal structure of L-tyrosine bound ThiH from Thermosinus carboxydivorans revealing an unusual protonation state of L-tyrosine upon binding. Structural comparison of ThiH with NosL and computational studies of the respective reactions they catalyze show that substrate activation is eased by tunneling effect and that subtle structural changes between the two enzymes affect, in particular, the hydrogen-atom abstraction by the 5´-deoxyadenosyl radical species, driving the difference in reaction specificity.


Assuntos
Liases , S-Adenosilmetionina , Catálise , S-Adenosilmetionina/metabolismo , Triptofano/metabolismo , Tirosina
2.
Oxid Med Cell Longev ; 2022: 1681623, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35464759

RESUMO

The immature germinal vesicle (GV) oocytes proceed through metaphase I (MI) division, extrude the first polar body, and become mature metaphase II (MII) oocytes for fertilization which is followed by preimplantation and postimplantation development until birth. Slc25a26 is the gene encoding S-adenosylmethionine carrier (SAMC), a member of the mitochondrial carrier family. Its major function is to catalyze the uptake of S-adenosylmethionine (SAM) from cytosol into mitochondria, which is the only known mitochondrial SAM transporter. In the present study, we demonstrated that excessive SLC25A26 accumulation in mouse oocytes mimicked naturally aged oocytes and resulted in lower oocyte quality with decreased maturation rate and increased reactive oxygen species (ROS) by impairing mitochondrial function. Increased level of Slc25a26 gene impacted gene expression in mouse oocytes such as mt-Cytb which regulates mitochondrial respiratory chain. Furthermore, increased level of Slc25a26 gene in fertilized oocytes slightly compromised blastocyst formation, and Slc25a26 knockout mice displayed embryonic lethality around 10.5 dpc. Taken together, our results showed that Slc25a26 gene plays a critical role in oocyte maturation and early mouse development.


Assuntos
Oócitos , S-Adenosilmetionina , Animais , Metáfase , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , S-Adenosilmetionina/metabolismo
3.
Mol Biol (Mosk) ; 56(2): 296-319, 2022.
Artigo em Russo | MEDLINE | ID: mdl-35403621

RESUMO

Methyltransferases (MTases) play an important role in the functioning of living systems, catalyzing the methylation reactions of DNA, RNA, proteins, and small molecules, including endogenous compounds and drugs. Many human diseases are associated with disturbances in the functioning of these enzymes; therefore, the study of MTases is an urgent and important task. Most MTases use the cofactor S-adenosyl-L-methionine (SAM) as a methyl group donor. SAM analogs are widely applicable in the study of MTases: they are used in studies of the catalytic activity of these enzymes, in identification of substrates of new MTases, and for modification of the substrates or substrate linking to MTases. In this review, new synthetic analogs of SAM and the problems that can be solved with their usage are discussed.


Assuntos
Metiltransferases , S-Adenosilmetionina , DNA/química , Humanos , Metionina , Metiltransferases/genética , Metiltransferases/metabolismo , RNA , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
4.
Cell Rep ; 39(2): 110672, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35417718

RESUMO

Phospholipid biosynthesis plays a role in mediating membrane-to-histone communication that influences metabolic decisions. Upon nutrient deprivation, phospholipid methylation generates a starvation signal in the form of S-adenosylmethionine (SAM) depletion, leading to dynamic changes in histone methylation. Here we show that the SAM-responsive methylation of H3K36 is critical for metabolic adaptation to nutrient starvation in the budding yeast Saccharomyces cerevisiae. We find that mutants deficient in H3K36 methylation exhibit defects in membrane integrity and pyrimidine metabolism and lose viability quickly under starvation. Adjusting the synthesis of phospholipids potently rewires metabolic pathways for nucleotide synthesis and boosts the production of antioxidants, ameliorating the defects resulting from the loss of H3K36 methylation. We further demonstrate that H3K36 methylation reciprocally regulates phospholipid synthesis by influencing redox balance. Our study illustrates an adaptive mechanism whereby phospholipid synthesis entails a histone modification to reprogram metabolism for adaptation in a eukaryotic model organism.


Assuntos
Histonas , Proteínas de Saccharomyces cerevisiae , Histonas/metabolismo , Metilação , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Sci Rep ; 12(1): 5593, 2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35379840

RESUMO

Tissue aging is a major cause of aging-related disabilities and a shortened life span. Understanding how tissue aging progresses and identifying the factors underlying tissue aging are crucial; however, the mechanism of tissue aging is not fully understood. Here we show that the biosynthesis of S-adenosyl-methionine (SAM), the major cellular donor of methyl group for methylation modifications, potently accelerates the aging-related defects during Drosophila oogenesis. An aging-related increase in the SAM-synthetase (Sam-S) levels in the germline leads to an increase in ovarian SAM levels. Sam-S-dependent biosynthesis of SAM controls aging-related defects in oogenesis through two mechanisms, decreasing the ability to maintain germline stem cells and accelerating the improper formation of egg chambers. Aging-related increases in SAM commonly occur in mouse reproductive tissue and the brain. Therefore, our results raise the possibility suggesting that SAM is the factor related to tissue aging beyond the species and tissues.


Assuntos
Drosophila , S-Adenosilmetionina , Envelhecimento , Animais , Metionina Adenosiltransferase , Camundongos , Oogênese
6.
Nucleic Acids Res ; 50(8): 4216-4245, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35412633

RESUMO

RNA methyltransferases (MTases) are ubiquitous enzymes whose hitherto low profile in medicinal chemistry, contrasts with the surging interest in RNA methylation, the arguably most important aspect of the new field of epitranscriptomics. As MTases become validated as drug targets in all major fields of biomedicine, the development of small molecule compounds as tools and inhibitors is picking up considerable momentum, in academia as well as in biotech. Here we discuss the development of small molecules for two related aspects of chemical biology. Firstly, derivates of the ubiquitous cofactor S-adenosyl-l-methionine (SAM) are being developed as bioconjugation tools for targeted transfer of functional groups and labels to increasingly visible targets. Secondly, SAM-derived compounds are being investigated for their ability to act as inhibitors of RNA MTases. Drug development is moving from derivatives of cosubstrates towards higher generation compounds that may address allosteric sites in addition to the catalytic centre. Progress in assay development and screening techniques from medicinal chemistry have led to recent breakthroughs, e.g. in addressing human enzymes targeted for their role in cancer. Spurred by the current pandemic, new inhibitors against coronaviral MTases have emerged at a spectacular rate, including a repurposed drug which is now in clinical trial.


Assuntos
Metiltransferases/antagonistas & inibidores , RNA , Desenvolvimento de Medicamentos , Humanos , S-Adenosilmetionina/análogos & derivados
7.
Methods Enzymol ; 666: 451-468, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35465927

RESUMO

Due to their biological importance and functional diversity, radical S-adenosylmethionine (rSAM) enzymes have become popular targets for electron paramagnetic resonance (EPR) spectroscopic studies. EPR spectroscopy is a powerful tool that allows for the observation of the iron-sulfur clusters as well as paramagnetic reaction intermediates, thus providing insight into their catalytic mechanisms. While the iron-sulfur clusters may be readily observable by EPR spectroscopy in the enzymes' resting states, radical intermediates are often elusive and must be trapped. Here, we describe a protocol for trapping and analyzing the Lys-Trp intermediate of the Lys-Trp-crosslinking rSAM enzyme SuiB, including modified expression and purification steps. This protocol is also intended to serve as a primer for trapping paramagnetic intermediates in other rSAM enzymes for studying by EPR spectroscopy.


Assuntos
Proteínas Ferro-Enxofre , S-Adenosilmetionina , Espectroscopia de Ressonância de Spin Eletrônica , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Enxofre/metabolismo
8.
Methods Enzymol ; 666: 469-487, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35465928

RESUMO

Nosiheptide is a ribosomally produced and post-translationally modified thiopeptide antibiotic that displays potent antibacterial activity in vitro, especially against Gram-positive pathogens. It comprises a core peptide macrocycle that contains multiple thiazole rings, dehydrated serine and threonine residues, a tri-substituted 3-hydroxypyridine ring and several other modifications. Among these additional modifications includes a 3,4-dimethyl-2-indolic acid (DMIA) moiety that bridges Glu6 and Cys8 of the core peptide to form a second smaller ring system. This side-ring system is formed by the action of NosN, a radical S-adenosylmethionine (SAM) enzyme that falls within the class C radical SAM methylase (RSMT) family. However, the true function of NosN is to transfer a methylene group from the methyl moiety of SAM to C4 of 3-methylindolic acid (MIA) attached in a thioester linkage to Cys8 of the core peptide to set up a highly electrophilic species. This species is then trapped by the side chain of Glu6, resulting in formation of a lactone and the side-ring system. The NosN reaction requires two simultaneously bound molecules of SAM. The first, SAMI, is cleaved to generate a 5'-deoxyadenosyl 5'-radical, which abstracts a hydrogen atom from the methyl group of the second molecule of SAM, SAMII. The resulting SAMII radical is believed to add to C4 of MIA, affording a radical intermediate on the MIA substrate. Herein we describe synthetic approaches that allow detection of this radical by electron paramagnetic resonance (EPR) spectroscopy.


Assuntos
Proteínas Ferro-Enxofre , S-Adenosilmetionina , Antibacterianos , Catálise , Proteínas Ferro-Enxofre/química , Metiltransferases/metabolismo , Peptídeos/química , S-Adenosilmetionina/metabolismo
9.
Molecules ; 27(7)2022 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-35408684

RESUMO

As a continuation of our earlier work against SARS-CoV-2, seven FDA-approved drugs were designated as the best SARS-CoV-2 nsp16-nsp10 2'-o-methyltransferase (2'OMTase) inhibitors through 3009 compounds. The in silico inhibitory potential of the examined compounds against SARS-CoV-2 nsp16-nsp10 2'-o-methyltransferase (PDB ID: (6W4H) was conducted through a multi-step screening approach. At the beginning, molecular fingerprints experiment with SAM (S-Adenosylmethionine), the co-crystallized ligand of the targeted enzyme, unveiled the resemblance of 147 drugs. Then, a structural similarity experiment recommended 26 compounds. Therefore, the 26 compounds were docked against 2'OMTase to reveal the potential inhibitory effect of seven promising compounds (Protirelin, (1187), Calcium folinate (1913), Raltegravir (1995), Regadenoson (2176), Ertapenem (2396), Methylergometrine (2532), and Thiamine pyrophosphate hydrochloride (2612)). Out of the docked ligands, Ertapenem (2396) showed an ideal binding mode like that of the co-crystallized ligand (SAM). It occupied all sub-pockets of the active site and bound the crucial amino acids. Accordingly, some MD simulation experiments (RMSD, RMSF, Rg, SASA, and H-bonding) have been conducted for the 2'OMTase-Ertapenem complex over 100 ns. The performed MD experiments verified the correct binding mode of Ertapenem against 2'OMTase exhibiting low energy and optimal dynamics. Finally, MM-PBSA studies indicated that Ertapenem bonded advantageously to the targeted protein with a free energy value of -43 KJ/mol. Furthermore, the binding free energy analysis revealed the essential amino acids of 2'OMTase that served positively to the binding. The achieved results bring hope to find a treatment for COVID-19 via in vitro and in vivo studies for the pointed compounds.


Assuntos
Metiltransferases , SARS-CoV-2 , Proteínas não Estruturais Virais , Proteínas Virais Reguladoras e Acessórias , Ertapenem/farmacologia , Ligantes , Metiltransferases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , S-Adenosilmetionina/química , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores
10.
Elife ; 112022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35438636

RESUMO

The redox reagent dithiothreitol (DTT) causes stress in the endoplasmic reticulum (ER) by disrupting its oxidative protein folding environment, which results in the accumulation and misfolding of the newly synthesized proteins. DTT may potentially impact cellular physiology by ER-independent mechanisms; however, such mechanisms remain poorly characterized. Using the nematode model Caenorhabditis elegans, here we show that DTT toxicity is modulated by the bacterial diet. Specifically, the dietary component vitamin B12 alleviates DTT toxicity in a methionine synthase-dependent manner. Using a forward genetic screen, we discover that loss-of-function of R08E5.3, an S-adenosylmethionine (SAM)-dependent methyltransferase, confers DTT resistance. DTT upregulates R08E5.3 expression and modulates the activity of the methionine-homocysteine cycle. Employing genetic and biochemical studies, we establish that DTT toxicity is a result of the depletion of SAM. Finally, we show that a functional IRE-1/XBP-1 unfolded protein response pathway is required to counteract toxicity at high, but not low, DTT concentrations.


Animal and plant cells synthesize a significant fraction of their proteins on a structure known as the endoplasmic reticulum. Researchers often use the molecule dithiothreitol to specifically target this compartment and learn more about its role. The toxin works by disturbing the complex chemical environment present in the reticulum, which is required for the proteins to assemble properly. However, it is important to clarify whether dithiothreitol could also affect other parts of the cell, as this could give rise to misleading results. To explore this possibility, Gokul G and Jogender Singh studied the effects of dithiothreitol on the millimetre-long roundworm Caenorhabditis elegans. Their experiments revealed that vitamin B12 could protect against dithiothreitol toxicity via a complex cascade of molecular events which reduced the levels of an important regulatory molecule known as S-adenosylmethionine. Crucially, the chemical reactions that dithiothreitol targeted took place outside the reticulum, suggesting that the toxin impairs processes in the wider cell. These results suggest that dithiothreitol should be reconsidered for use in endoplasmic reticulum studies. However, they also imply that this toxin could be beneficial in small doses, as a reduced concentration of S-adenosylmethionine increases lifespan and health in a variety of organisms.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Ditiotreitol/metabolismo , Ditiotreitol/toxicidade , Retículo Endoplasmático/metabolismo , Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo
11.
J Med Chem ; 65(8): 6231-6249, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35439007

RESUMO

Enzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure-activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔTm ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/tratamento farmacológico , COVID-19/virologia , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/química , Humanos , Metiltransferases , Simulação de Acoplamento Molecular , RNA Viral/genética , S-Adenosilmetionina , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química
12.
Commun Biol ; 5(1): 313, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35383287

RESUMO

The global dietary supplement market is valued at over USD 100 billion. One popular dietary supplement, S-adenosylmethionine, is marketed to improve joints, liver health and emotional well-being in the US since 1999, and has been a prescription drug in Europe to treat depression and arthritis since 1975, but recent studies questioned its efficacy. In our body, S-adenosylmethionine is critical for the methylation of nucleic acids, proteins and many other targets. The marketing of SAM implies that more S-adenosylmethionine is better since it would stimulate methylations and improve health. Previously, we have shown that methylation reactions regulate biological rhythms in many organisms. Here, using biological rhythms to assess the effects of exogenous S-adenosylmethionine, we reveal that excess S-adenosylmethionine disrupts rhythms and, rather than promoting methylation, is catabolized to adenine and methylthioadenosine, toxic methylation inhibitors. These findings further our understanding of methyl metabolism and question the safety of S-adenosylmethionine as a supplement.


Assuntos
Adenina , S-Adenosilmetionina , Suplementos Nutricionais , Fígado/metabolismo , Metilação , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/farmacologia
13.
Acc Chem Res ; 55(9): 1249-1261, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35420432

RESUMO

The central dogma of molecular biology hinges on messenger RNA (mRNA), which presents a blueprint of the genetic information encoded in the DNA and serves as a template for translation into proteins. In addition to its fundamental importance in basic research, this class of biomolecules has recently become the first approved Covid vaccine, underscoring its utility in medical applications.Eukaryotic mRNA is heavily processed, including the 5' cap as the primary hallmark. This 5' cap protects mRNA from degradation by exoribonucleases but also interacts specifically with several proteins and enzymes to ensure mRNA turnover and processing, like splicing, export from the nucleus to the cytoplasm, and initiation of translation. The absence of a 5' cap leads to a strong immune response, and the methylation status contributes to distinguishing self from non-self RNA.Non-natural modifications of the 5' cap provide an avenue to label mRNAs and make them accessible to analyses, which is important to study their cellular localization, trafficking, and binding partners. They bear potential to engineer mRNAs, e.g., more stable or immunogenic mRNAs that are still translated, by impacting select interactions in a distinct manner. The modification of the 5' cap itself is powerful as it can be applied to make long mRNAs (∼1000 nt, not directly accessible by solid-phase synthesis) by in vitro transcription.This Account describes our contribution to the field of chemo-enzymatic modification of mRNA at the 5' cap. Our approach relies on RNA methyltransferases (MTases) with promiscuous activity on analogues of their natural cosubstrate S-adenosyl-L-methionine (AdoMet). We will describe how RNA MTases in combination with non-natural cosubstrates provide access to site-specific modification of different positions of the 5' cap, namely, the N2 and N7 position of guanosine and the N6 position of adenosine as the transcription start nucleotide (TSN) and exemplify strategies to make long mRNAs with modified 5' caps.We will compare the chemical and enzymatic synthesis of the AdoMet analogues used for this purpose. We could overcome previous limitations in methionine adenosyltransferase (MAT) substrate scope by engineering variants (termed PC-MATs) with the ability to convert methionine analogues with benzylic and photocaging groups at the sulfonium ion.The final part of this Account will highlight applications of the modified mRNAs. Like in many chemo-enzymatic approaches, a versatile strategy is to install small functional groups enzymatically and use them as handles in subsequent bioorthogonal reactions. We showed fluorescent labeling of mRNAs via different types of click chemistry in vitro and in cells. In a second line of applications, we used the handles to make mRNAs amenable for analyses, most notably next-generation sequencing. In the case of extremely promiscuous enzymes, the direct installation of photo-cross-linking groups was successful also and provided a way to covalently bind protein-interaction partners. Finally, the non-natural modifications of mRNAs can also modulate the properties of mRNAs. Propargylation of Am as the transcription start nucleotide at its N6 position maintained the translation of mRNAs but increased their immunogenicity. The installation of photocaging groups provides a way to revert these effects and control interactions by light.


Assuntos
RNA Mensageiro , S-Adenosilmetionina , Vacinas contra COVID-19 , Humanos , Metionina , Metiltransferases/genética , Metiltransferases/metabolismo , Nucleotídeos , RNA , RNA Mensageiro/metabolismo , S-Adenosilmetionina/química
14.
Behav Brain Res ; 427: 113866, 2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35367299

RESUMO

Epigenetic changes are an important pathogenic mechanism in many diseases, including a variety of psychiatric disorders such as Autism Spectrum Disorder (ASD) and depression. Methyl donors such as S-Adenosyl-Methionine (SAMe) may cause epigenetic modifications, especially during embryonic development when the epigenetic memory is established. We treated pregnant submissive (Sub) mice exhibiting depressive-like phenotype with SAMe during days 12-14 of gestation aiming to alleviate the depressive - like symptoms in their offspring and normalize the expression in their prefrontal cortex of several genes possibly involved in depression. We also aimed to define possible gender differences of the effects of SAMe on the measured parameters. Treatment of the Dams with SAMe did not affect the early neurodevelopmental milestones in males or females. The results of the behavioral tests showed improvement in some behavioral parameters compared to saline treated Sub mice. Several of these improvements were gender related. Prenatal SAMe treatment mainly improved sociability, as observed in the three chambers social interaction test, in both genders. It also improved the increased locomotion (as observed by the open field test) in the female mice, but not in males. Prenatal SAMe increased the expression of Vegfa and Flt1 in males, but not in females. The expression of IgfII and SynIIb increased in males and decreased in females and the expression of serotonin receptor Htr2A did not change in both genders. In our mouse model of depression, prenatal treatment with SAMe significantly improved some parameters of depressive like behavior and normalized the expression of several genes related to depression. The gender differences observed in our studies may explain the sex related differences in the clinical presentation of depression and the different gender related response to treatment.


Assuntos
Transtorno do Espectro Autista , Efeitos Tardios da Exposição Pré-Natal , Animais , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Comportamento Animal , Modelos Animais de Doenças , Feminino , Expressão Gênica , Hierarquia Social , Humanos , Masculino , Camundongos , Gravidez , S-Adenosilmetionina/farmacologia
15.
J Med Chem ; 65(6): 4600-4615, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35293760

RESUMO

Inhibition of the S-adenosyl methionine (SAM)-producing metabolic enzyme, methionine adenosyltransferase 2A (MAT2A), has received significant interest in the field of medicinal chemistry due to its implication as a synthetic lethal target in cancers with the deletion of the methylthioadenosine phosphorylase (MTAP) gene. Here, we report the identification of novel MAT2A inhibitors with distinct in vivo properties that may enhance their utility in treating patients. Following a high-throughput screening, we successfully applied the structure-based design lessons from our first-in-class MAT2A inhibitor, AG-270, to rapidly redesign and optimize our initial hit into two new lead compounds: a brain-penetrant compound, AGI-41998, and a potent, but limited brain-penetrant compound, AGI-43192. We hope that the identification and first disclosure of brain-penetrant MAT2A inhibitors will create new opportunities to explore the potential therapeutic effects of SAM modulation in the central nervous system (CNS).


Assuntos
Metionina Adenosiltransferase , Neoplasias , Encéfalo/metabolismo , Desenho de Fármacos , Humanos , Neoplasias/tratamento farmacológico , S-Adenosilmetionina/metabolismo
16.
J Am Chem Soc ; 144(11): 5087-5098, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35258967

RESUMO

Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe-4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g∥ = 2.035 and g⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at giso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5'-deoxyadenosyl radical (5'-dAdo•). The time course of the 5'-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5'-dAdo• by Fe-C5' bond homolysis, and the 5'-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5'-dAdo• intermediate trapped just prior to reaction with the substrate.


Assuntos
Metionina , S-Adenosilmetionina , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , S-Adenosilmetionina/metabolismo
17.
J Am Chem Soc ; 144(13): 5673-5684, 2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35344653

RESUMO

Cobalamin (Cbl)-dependent S-adenosyl-l-methionine (AdoMet) radical methylases are known for their use of a dual cofactor system to perform challenging radical methylation reactions at unactivated carbon and phosphorus centers. These enzymes are part of a larger subgroup of Cbl-dependent AdoMet radical enzymes that also perform difficult ring contractions and radical rearrangements. This subgroup is a largely untapped reservoir of diverse chemistry that requires steady efforts in biochemical and structural characterization to reveal its complexity. In this Perspective, we highlight the significant efforts over many years to elucidate the function, mechanism, and structure of TsrM, an unexpected nonradical methylase in this subgroup. We also discuss recent achievements in characterizing radical methylase subgroup members that exemplify how key tools in mechanistic enzymology are valuable time and again. Finally, we identify recent enzyme activity studies that have made use of bioinformatic analyses to expand our definition of the subgroup. Additional breakthroughs in radical (and nonradical) enzymatic chemistry and challenging transformations from the unexplored space of this subgroup are undoubtedly on the horizon.


Assuntos
S-Adenosilmetionina , Vitamina B 12 , Metionina , Metilação , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/química
18.
J Am Chem Soc ; 144(10): 4478-4486, 2022 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-35238201

RESUMO

The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.


Assuntos
Desidratação , S-Adenosilmetionina , Adenosilmetionina Descarboxilase , Humanos , Nucleosídeos , S-Adenosilmetionina/química
19.
Mol Cell ; 82(9): 1631-1642.e6, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35316659

RESUMO

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas , Antivirais/farmacologia , Imunidade Inata , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , S-Adenosilmetionina , Replicação Viral
20.
Chem Commun (Camb) ; 58(16): 2738-2741, 2022 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-35118483

RESUMO

B12-Independent glycerol dehydratase (GD) is a glycyl radical enzyme in the biotransformation of glycerol to 1,3-propanediol. GD requires the activating enzyme GD-AE to initiate the radical reaction. GD-AE belongs to the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. However, a previous study showed that GD-AE cleaves SAM unconventionally to generate 5'-deoxy-5'-methylthioadenosine. Herein, we show that GD-AE actually cleaves SAM to form 5'-deoxyadenosine, similar to other radical SAM enzymes. Furthermore, with the synthesized glycerol analogue 2-deoxy-2-fluoroglycerol, we demonstrate that B12-independent GD catalyzes the glycerol dehydration reaction by direct elimination of the C-2 hydroxyl group of a ketyl radical rather than 1,2-OH migration.


Assuntos
Hidroliases/metabolismo , Vitamina B 12/metabolismo , Biocatálise , Desoxiadenosinas/química , Desoxiadenosinas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Estrutura Molecular , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
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