RESUMEN

Methionine, an indispensable amino acid crucial for dietary balance, intricately governs metabolic pathways. Disruption in its equilibrium has the potential to heighten homocysteine levels in both plasma and tissues, posing a conceivable risk of inducing inflammation and detriment to the integrity of vascular endothelial cells. The intricate interplay between methionine metabolism, with a specific focus on S-adenosyl-L-methionine (SAM), and the onset of thoracic aortic dissection (TAD) remains enigmatic despite acknowledging the pivotal role of inflammation in this vascular condition. In an established murine model induced by ß-aminopropionitrile monofumarate (BAPN), we delved into the repercussions of supplementing with S-adenosyl-L-methionine (SAM) on the progression of TAD. Our observations uncovered a noteworthy improvement in aortic dissection and rupture rates, accompanied by a marked reduction in mortality upon SAM supplementation. Notably, SAM supplementation exhibited a considerable protective effect against BAPN-induced degradation of elastin and the extracellular matrix. Furthermore, SAM supplementation demonstrated a robust inhibitory influence on the infiltration of immune cells, particularly neutrophils and macrophages. It also manifested a notable reduction in the inflammatory polarization of macrophages, evident through diminished accumulation of MHC-IIhigh macrophages and reduced expression of inflammatory cytokines such as IL1ß and TNFα in macrophages. Simultaneously, SAM supplementation exerted a suppressive effect on the activation of CD4 + and CD8 + T cells within the aorta. This was evidenced by an elevated proportion of CD44- CD62L + naïve T cells and a concurrent decrease in CD44 + CD62L- effector T cells. In summary, our findings strongly suggest that the supplementation of SAM exhibits remarkable efficacy in alleviating BAPN-induced aortic inflammation, consequently impeding the progression of thoracic aortic dissection.

RESUMEN

S-Adenosyl-l-methionine (SAM)-mediated methylation of biomolecules controls their function and regulates numerous vital intracellular processes. Analogs of SAM with a reporter group in place of the S-methyl group are widely used to study these processes. However, many of these analogs are chemically unstable that largely limits their practical application. We have developed a new compound, SAM-P H , which contains an H-phosphinic group (-P(O)(H)OH) instead of the SAM carboxylic group. SAM-P H is significantly more stable than SAM, retains functional activity in catechol-O-methyltransferase and methyltransferase WBSCR27 reactions. The last is associated with Williams-Beuren syndrome. Rac-SAM-P H was synthesized chemically, while (R,S)-SAM-P H and its analogs were prepared enzymatically either from H-phosphinic analogs of methionine (Met-PH) or H-phosphinic analog of S-adenosyl-l-homocysteine (SAH-P H ) using methionine adenosyltransferase 2A or halide methyltransferases, respectively. SAH-P H undergoes glycoside bond cleavage in the presence of methylthioadenosine nucleosidase like natural SAH. Thus, SAM-P H and its analogs are promising new tools for investigating methyltransferases and incorporating reporter groups into their substrates.

RESUMEN

DNA methylation, an important epigenetic modification, is catalyzed by DNA methyltransferases and is essential in the regulation of gene expression. Here, the utility of an N-mustard analog designed to mimic the native methyl donor, S-adenosyl-L-methionine (SAM), was explored with the DNA methyltransferase 3A catalytic domain (DNMT3AC). In lieu of the expected analog transfer to DNA, methyltransferase activity was instead inhibited in a concentration dependent manner. Further investigation into the mechanism of analog inhibition did not reveal a typical competitive mechanism. Instead, mass spectrometry analysis provided direct evidence of two cysteine residues in the SAM binding site covalently modified by the SAM analog and confirmed its' function as an irreversible inhibitor of DNMT3AC.

RESUMEN

DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-l-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-l-homocysteine (SAH) into L-homocysteine. As a result, the SAM-to-SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts, but there has yet to be a study looking at the potential downstream impact of DZNep treatment on proteins other than histones. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small-molecule inhibitors. In addition to ligand binding, post-translational modifications and protein-protein interactions impact thermal stability. Here, we sought to characterize the protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in the SAM/SAH levels. This study systematically evaluates DZNep's impact on the transcriptome, the proteome, and the thermal stability of proteins.

Asunto(s)

Adenosina , Estabilidad Proteica , Humanos , Células HEK293 , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/química , Estabilidad Proteica/efectos de los fármacos , Metilación , Adenosilhomocisteinasa/antagonistas & inhibidores , Adenosilhomocisteinasa/metabolismo , Temperatura

RESUMEN

Cellular senescence significantly affects the proliferative and differentiation capacities of mesenchymal stem cells (MSCs). Identifying key regulators of senescence and exploring potential intervention strategies, including drug-based approaches, are active areas of research. In this context, S-adenosyl-l-methionine (SAM), a critical intermediate in sulfur amino acid metabolism, emerges as a promising candidate for mitigating MSC senescence. In a hydrogen peroxide-induced MSC aging model (100 µM for 2 hours), SAM (50 and 100 µM) was revealed to alleviate the senescence of MSCs, and also attenuated the level of reactive oxygen species and enhanced the adipogenic and osteogenic differentiation in senescent MSCs. In a premature aging mouse model (subcutaneously injected with 150 mg/kg/day d-galactose in the neck and back for 7 weeks), SAM (30 mg/kg/day by gavage for 5 weeks) was shown to delay the overall aging process while increasing the number and thickness of bone trabeculae in the distal femur. Mechanistically, activation of PI3K/AKT signaling and increased phosphorylation of forkhead box O3 (FOXO3a) was proved to be associated with the antisenescence role of SAM. These findings highlight that the PI3K/AKT/FOXO3a axis in MSCs could play a crucial role in MSCs senescence and suggest that SAM may be a potential therapeutic drug for MSCs senescence and related diseases.

Asunto(s)

Senescencia Celular , Proteína Forkhead Box O3 , Células Madre Mesenquimatosas , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , S-Adenosilmetionina , Transducción de Señal , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Animales , Senescencia Celular/efectos de los fármacos , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , Transducción de Señal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , S-Adenosilmetionina/farmacología , S-Adenosilmetionina/metabolismo , Ratones , Diferenciación Celular/efectos de los fármacos , Masculino , Humanos , Ratones Endogámicos C57BL

RESUMEN

Objective: The present study was designed to investigate the nephroprotective and immunoprotective effects of S-adenosyl-L-methionine (SAMe) in comparison to N-acetylcysteine (NAC) against ochratoxin A (OTA) - intoxication. Methods: Forty-eight adult male Sprague-Dawley rats were categorized into four groups: Control; OTA intoxication (5 mg OTA/kg diet); OTA + NAC, rats received 200 mg NAC/day before feeding balanced diet contaminated with OTA; and (OTA + SAMe). Rats received 200 mg SAMe/day dissolved in distilled water orally just before feeding a balanced diet contaminated with OTA. Results: OTA administration altered serum kidney function biomarkers. These effects were pronouncedly alleviated by treatment with NAC. Results revealed a correlation between OTA-induced immunotoxicity and the reduced white blood cell (WBC) count. Treatments with SAMe significantly improved the WBCs count and hemoglobin concentration. Conclusion: NAC and SAMe have a protective role against nephrotoxicity and immunotoxicity induced by continuous administration of OTA. NAC was more effective in reducing OTA nephrotoxicity, whereas SAMe was more potent than NAC in reducing OTA immunotoxicity.

RESUMEN

S-Adenosyl-L-methionine (SAM) is a substrate for many enzyme-catalyzed reactions and provides methyl groups in numerous biological methylations, and thus has vast applications in the agriculture and medical field. Saccharomyces cerevisiae has been engineered as a platform with significant potential for producing SAM, but the current production has room for improvement. Thus, a method that consists of a series of metabolic engineering strategies was established in this study. These strategies included enhancing SAM synthesis, increasing ATP supply, down-regulating SAM metabolism, and down-regulating competing pathway. After combinatorial metabolic engineering, Bayesian optimization was conducted on the obtained strain C262P6S to optimize the fermentation medium. A final yield of 2972.8 mg·L-1 at 36 h with 29.7% of the L-Met conversion rate in the shake flask was achieved, which was 26.3 times higher than that of its parent strain and the highest reported production in the shake flask to date. This paper establishes a feasible foundation for the construction of SAM-producing strains using metabolic engineering strategies and demonstrates the effectiveness of Bayesian optimization in optimizing fermentation medium to enhance the generation of SAM.

Asunto(s)

Metionina , S-Adenosilmetionina , Metionina/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ingeniería Metabólica/métodos , Teorema de Bayes , Fermentación , Racemetionina/metabolismo

RESUMEN

This systematic review and meta-analysis aimed to assess the efficacy and acceptability of S-adenosyl-L-methionine (SAMe) in treating depression. We conducted a comprehensive search of PubMed, Embase, Cochrane Library, and ClinialTrials.gov from inception to July 3, 2023, identifying randomized controlled trials comparing SAMe with placebo or antidepressants (ADs). We synthesized data on reduced depressive symptoms (efficacy) and overall dropout rates (acceptability) using a random-effects model for pairwise frequentist meta-analysis. Our analysis included 23 trials (N = 2183) classified into three categories: 11 trials comparing SAMe and placebo, 5 trials comparing SAMe plus ADs and placebo plus ADs, and 7 trials comparing SAMe and ADs. Differences between experimental and control interventions in reducing depressive symptoms were observed: i) SAMe demonstrated significantly superior efficacy compared to placebo (SMD = -0.58, 95% CI = -0.93 to -0.23, I2 = 68%); ii) in conjunction with ADs, SAMe did not show a significant difference from placebo (SMD = -0.22, 95%CI = -0.63 to 0.19, I2 = 76%); and iii) SAMe did not exhibit a significant difference from ADs alone (SMD = 0.06, 95%CI = -0.06 to 0.18, I2 = 49%). No significant differences in dropout rates were observed across the three comparison categories. Moderate-certainty evidence suggests that SAMe monotherapy may offer a moderate therapeutic benefit in alleviating depressive symptoms. Considering its favorable acceptability profile, SAMe monotherapy should be considered as a treatment option for patients with depression. However, uncertainties regarding its efficacy as an adjunct to AD and its comparative efficacy with ADs remain unresolved.

Asunto(s)

Antidepresivos , S-Adenosilmetionina , Humanos , S-Adenosilmetionina/uso terapéutico , Antidepresivos/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como Asunto , Depresión/tratamiento farmacológico , Depresión/psicología , Resultado del Tratamiento

RESUMEN

BACKGROUND: Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. RESULTS: Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in ß-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. CONCLUSIONS: Two novel P. pastoris chassis hosts with impaired ß-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.

Asunto(s)

Metanol , Pichia , Saccharomycetales , Humanos , Pichia/metabolismo , Metanol/metabolismo , Glicerol/metabolismo , Adenosina Trifosfato/metabolismo , Carbono/metabolismo , Pared Celular/metabolismo , Polisacáridos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Methylated natural products are widely spread in nature. S-Adenosyl-l-methionine (SAM) is the secondary abundant cofactor and the primary methyl donor, which confer natural products with structural and functional diversification. The increasing demand for SAM-dependent natural products (SdNPs) has motivated the development of microbial cell factories (MCFs) for sustainable and efficient SdNP production. Insufficient and unsustainable SAM availability hinders the improvement of SdNP MCF performance. From the perspective of developing MCF, this review summarized recent understanding of de novo SAM biosynthesis and its regulatory mechanism. SAM is just the methyl mediator but not the original methyl source. Effective and sustainable methyl source supply is critical for efficient SdNP production. We compared and discussed the innate and relatively less explored alternative methyl sources and identified the one involving cheap one-carbon compound as more promising. The SAM biosynthesis is synergistically regulated on multilevels and is tightly connected with ATP and NAD(P)H pools. We also covered the recent advancement of metabolic engineering in improving intracellular SAM availability and SdNP production. Dynamic regulation is a promising strategy to achieve accurate and dynamic fine-tuning of intracellular SAM pool size. Finally, we discussed the design and engineering constraints underlying construction of SAM-responsive genetic circuits and envisioned their future applications in developing SdNP MCFs.

Asunto(s)

Productos Biológicos , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Ingeniería Metabólica

RESUMEN

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm-1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.

Asunto(s)

Metionina , S-Adenosilmetionina , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Sales (Química) , Metiltransferasas/química , Metiltransferasas/metabolismo , Racemetionina , Isótopos

RESUMEN

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum.

Asunto(s)

Criptosporidiosis , Cryptosporidium , Humanos , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/química , Metionina Adenosiltransferasa/metabolismo , S-Adenosilmetionina/metabolismo , Proyectos Piloto , Cryptosporidium/metabolismo , Escherichia coli/genética

RESUMEN

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

Asunto(s)

Desoxiadenosinas , Metionina Adenosiltransferasa , Neoplasias , Proteína-Arginina N-Metiltransferasas , Purina-Nucleósido Fosforilasa , S-Adenosilmetionina , Animales , Humanos , Ratones , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxiadenosinas/antagonistas & inhibidores , Desoxiadenosinas/genética , Desoxiadenosinas/metabolismo , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Metionina Adenosiltransferasa/antagonistas & inhibidores , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Neoplasias/genética , Neoplasias/fisiopatología , Neoplasias/terapia , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/metabolismo , Pirrolidinas/farmacología , Pirrolidinas/uso terapéutico , S-Adenosilmetionina/metabolismo

RESUMEN

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616-19-5 was successfully obtained and exhibited higher SAM production (0.41 g/L vs 1.39 g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616-19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616-19-5 could produce 10.92 ± 0.2 g/L SAM in a 5-L fermenter after 96 h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production.

Asunto(s)

Metionina , S-Adenosilmetionina , Saccharomyces cerevisiae/genética , Ensayos Analíticos de Alto Rendimiento , Fitomejoramiento , Racemetionina

RESUMEN

The switching on or off of methylation, a change from a normal methylation to hyper or hypo methylation is implicated in many diseases that include cancers, infectious, neurodegenerative diseases and others. Methyltransferases are one of the most sought targets that have diversified for the methylation of a variety of substrates. However, without S-adenosyl-l-methionine (SAM), the universal methyl donor, the majority of the methyltransferases remain functionally inactive. In this article, we did a comprehensive analysis of all available SAM-receptor crystal structures at atom, moiety and structure levels to gain deeper insights into the structure and function of SAM. SAM demonstrated flexibility in binding to a variety of receptors irrespective of the size of the binding pockets. Further analysis of the binding pockets resulted in all SAM conformations clustering into four natural shapes. The conserved interaction analysis provides an unambiguous orientation of SAM binding to receptors which has been elusive till now. SAM peptide moiety (SPM) and SAM nucleobase moiety (SNM) show up to 89% interactions with receptors whereas only 11% interactions with SAM ribose moiety (SRM). It is found that SPM and SNM terminal atoms anchor to the highly conserved receptor subsites creating a workbench for catalysis. It is seen that every interacting atom and its position is crucial in the methyl transfer phenomenon. A very unique observation is that the methyl group of SAM does not have even one interaction with the receptor. The deep insights gained help in the design and development of novel drugs against the methyltransferases.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Metiltransferasas , S-Adenosilmetionina , Metiltransferasas/química , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Metilación , Catálisis

RESUMEN

Enzymes of the radical S-adenosyl-l-methionine (radical SAM, RS) superfamily, the largest in nature, catalyze remarkably diverse reactions initiated by H-atom abstraction. Glycyl radical enzyme activating enzymes (GRE-AEs) are a growing class of RS enzymes that generate the catalytically essential glycyl radical of GREs, which in turn catalyze essential reactions in anaerobic metabolism. Here, we probe the reaction of the GRE-AE pyruvate formate-lyase activating enzyme (PFL-AE) with the peptide substrate RVSG734YAV, which mimics the site of glycyl radical formation on the native substrate, pyruvate formate-lyase. Time-resolved freeze-quench electron paramagnetic resonance spectroscopy shows that at short mixing times reduced PFL-AE + SAM reacts with RVSG734YAV to form the central organometallic intermediate, Ω, in which the adenosyl 5'C is covalently bound to the unique iron of the [4Fe-4S] cluster. Freeze-trapping the reaction at longer times reveals the formation of the peptide G734• glycyl radical product. Of central importance, freeze-quenching at intermediate times reveals that the conversion of Ω to peptide glycyl radical is not concerted. Instead, homolysis of the Ω Fe-C5' bond generates the nominally "free" 5'-dAdo• radical, which is captured here by freeze-trapping. During cryoannealing at 77 K, the 5'-dAdo• directly abstracts an H-atom from the peptide to generate the G734• peptide radical trapped in the PFL-AE active site. These observations reveal the 5'-dAdo• radical to be a well-defined intermediate, caught in the act of substrate H-atom abstraction, providing new insights into the mechanistic steps of radical initiation by RS enzymes.

Asunto(s)

Proteínas Hierro-Azufre , S-Adenosilmetionina , S-Adenosilmetionina/química , Acetiltransferasas/metabolismo , Metionina , Espectroscopía de Resonancia por Spin del Electrón , Péptidos/metabolismo , Proteínas Hierro-Azufre/metabolismo

RESUMEN

Methylation plays a vital role in biological systems. SAM (S-adenosyl-L-methionine), an abundant cofactor in life, acts as a methyl donor in most biological methylation reactions. SAM-dependent methyltransferases (MTase) transfer a methyl group from SAM to substrates, thereby altering their physicochemical properties or biological activities. In recent years, many SAM analogues with alternative methyl substituents have been synthesized and applied to methyltransferases that specifically transfer different groups to the substrates. These include functional groups for labeling experiments and novel alkyl modifications. This review summarizes the recent progress in the synthesis and application of SAM methyl analogues and prospects for future research directions in this field.

Asunto(s)

Metionina , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Metiltransferasas/metabolismo , Metilación , Racemetionina

RESUMEN

S-adenosyl- l-methionine (SAM) is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, an SAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

Asunto(s)

Riboswitch , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ensayos Analíticos de Alto Rendimiento , Riboswitch/genética , Fermentación

RESUMEN

Introduction: metabolic syndrome comprises a combination of diabetes, high blood pressure, and obesity, and metabolic associated fatty liver disease (MAFLD) is associated with it. Objective: to evaluate the effect of supplementation with S-adenosyl-L-methionine + N-acetylcysteine + thioctic acid + vitamin B6 (MetioNac®) for 3 months on lipidic and biochemical parameters in subjects with metabolic syndrome and at risk of MAFLD. The reduction in body weight and the oxidative stress markers malondialdehyde (MDA) and superoxide dismutase (SOD) were also evaluated. Methods: patients with metabolic syndrome, at risk of MAFLD (FIB-4 < 1.30), and with an indication for weight reduction were recruited (n = 15). Control group followed a semipersonalized Mediterranean diet (MD) for weight reduction, according to the recommendations of the Spanish Society for the Study of Obesity (SEEDO). Experimental group, in addition to the MD, took three capsules of MetioNac® supplement per day. Results: compared with the control group, subjects taking MetioNac® showed significant (p < 0.05) reductions in the levels of TG and VLDL-c, as well as in total cholesterol, LDL-c, and glucose levels. They also showed increased levels of HDL-c. Levels of AST and ALT decreased after the intervention with MetioNac®, but this decrease did not reach statistical significance. Weight loss was observed in both groups. Conclusion: supplementation with MetioNac® may be protective against hyperlipidemia, insulin resistance, and overweight among metabolic syndrome patients. Further studies on this issue are needed in a larger population.(AU)

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Introducción: el síndrome metabólico se define como una combinación de diabetes, hipertensión arterial y obesidad, que se asocia con laenfermedad del hígado graso asociada a disfunción metabólica.Objetivo: evaluar el efecto de la suplementación con S-adenosil-L-metionina + N-acetilcisteína + ácido tióctico + vitamina B6 (MetioNac®)durante 3 meses sobre parámetros lipídicos y bioquímicos en sujetos con síndrome metabólico y en riesgo de enfermedad del hígado grasoasociada a disfunción metabólica. También se evaluaron la reducción del peso corporal y los marcadores de estrés oxidativo malondialdehído(MDA) y superóxido dismutasa (SOD).Métodos: se reclutaron pacientes con síndrome metabólico, riesgo de enfermedad del hígado graso asociada a disfunción metabólica (FIB-4 < 1,30) y con indicación de reducción de peso (n = 15). El grupo control siguió una dieta mediterránea (DM) semipersonalizada para la reducciónde peso, de acuerdo con las recomendaciones de la Sociedad Española para el Estudio de la Obesidad (SEEDO). El grupo intervención, ademásde la DM, tomó tres cápsulas diarias de MetioNac®.Resultados: en comparación con el grupo de control, los sujetos que tomaron MetioNac® mostraron reducciones significativas (p < 0.05) en losniveles de TG y VLDL-c, así como en los niveles de colesterol total, LDL-c y glucosa. También mostraron niveles elevados de HDL-c. Los nivelesde AST y ALT disminuyeron después de la intervención con MetioNac®, pero esta disminución no fue estadísticamente significativa. También seobservó una pérdida de peso en ambos grupos.Conclusión: la suplementación con MetioNac® puede proteger contra la hiperlipidemia, la insulinorresistencia y el sobrepeso en pacientes consíndrome metabólico. Sin embargo, es necesario realizar más estudios y seleccionar un mayor número de participantes.(AU)

Asunto(s)

Humanos , Masculino , Femenino , Hígado Graso/complicaciones , Síndrome Metabólico , Ácido Tióctico/administración & dosificación , S-Adenosilmetionina/administración & dosificación , Acetilcisteína , Vitamina B 6 , 52503 , Nutrición, Alimentación y Dieta , Suplementos Dietéticos , Estudios de Casos y Controles

RESUMEN

Komagataella phaffii, formerly Pichia pastoris (P. pastoris), is a promising methylotrophic yeast used in industry to produce recombinant protein and valuable metabolites. In this study, a genome-scale metabolic model (GEMs) was reconstructed and used to assess P. pastoris' metabolic capabilities for the production of S-adenosyl-L-methionine (AdoMet or SAM or SAMe) from individual carbon sources along with the addition of L-methionine. In a model-driven P. pastoris strain, the well-established genome-scale metabolic model iAUKM can be implemented to predict high valuable metabolite production. The model, iAUKM, was created by merging the previously published iMT1026 model and the draught model generated using Raven toolbox from the KEGG database which covered 2309 enzymatic reactions associated with 1033 metabolic genes and 1750 metabolites. The highly curated model was successful in capturing P. pastoris growth on various carbon sources, as well as AdoMet production under various growth conditions. Many overexpression gene targets for increasing AdoMet accumulation in the cell have been predicted for various carbon sources. Inorganic phosphatase (IPP) was one of the predicted overexpression targets as revealed from simulations using iAUKM. When IPP gene was integrated into P. pastoris, we found that AdoMet accumulation increased by 16% and 14% using glucose and glycerol as carbon sources, respectively. Our in silico results shed light on the factors limiting AdoMet production, as well as key pathways for rationalized engineering to increase AdoMet yield.

Asunto(s)

Metionina , S-Adenosilmetionina , Racemetionina , Carbono