S-adenosylmethionine Concentrations Research Articles

Enhanced Bacitracin Production by Systematically Engineering S-Adenosylmethionine Supply Modules in Bacillus licheniformis.

Bacitracin is a broad-spectrum veterinary antibiotic that widely used in the fields of veterinary drug and feed additive. S-Adenosylmethionine (SAM) is a critical factor involved in many biochemical reactions, especially antibiotic production. However, whether SAM affects bacitracin synthesis is still unknown. Here, we want to analyze the relationship between SAM supply and bacitracin synthesis, and then metabolic engineering of SAM synthetic pathway for bacitracin production in Bacillus licheniformis. Firstly, our results implied that SAM exogenous addition benefited bacitracin production, which yield was increased by 12.13% under the condition of 40 mg/L SAM addition. Then, SAM synthetases and Methionine (Met) synthetases from B. licheniformis, Corynebacterium glutamicum, and Saccharomyces cerevisiae were screened and overexpressed to improve SAM accumulation, and the combination of SAM synthetase from S. cerevisiae and Met synthetase from B. licheniformis showed the best performance, and 70.12% increase of intracellular SAM concentration (31.54 mg/L) and 13.08% increase of bacitraicn yield (839.54 U/mL) were achieved in resultant strain DW2-KE. Furthermore, Met transporters MetN and MetP were, respectively, identified as Met exporter and importer, and bacitracin yield was further increased by 5.94% to 889.42 U/mL via deleting metN and overexpressing metP in DW2-KE, attaining strain DW2-KENP. Finally, SAM nucleosidase gene mtnN and SAM decarboxylase gene speD were deleted to block SAM degradation pathways, and bacitracin yield of resultant strain DW2-KENPND reached 957.53 U/mL, increased by 28.97% compared to DW2. Collectively, this study demonstrated that SAM supply served as the critical role in bacitracin synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin.

The Balance Metabolism Safety Net: Integration of Stress Signals by Interacting Transcriptional Factors in Streptomyces and Related Actinobacteria.

Soil dwelling Streptomyces species are faced with large variations in carbon or nitrogen sources, phosphate, oxygen, iron, sulfur, and other nutrients. These drastic changes in key nutrients result in an unbalanced metabolism that have undesirable consequences for growth, cell differentiation, reproduction, and secondary metabolites biosynthesis. In the last decades evidence has accumulated indicating that mechanisms to correct metabolic unbalances in Streptomyces species take place at the transcriptional level, mediated by different transcriptional factors. For example, the master regulator PhoP and the large SARP-type regulator AfsR bind to overlapping sequences in the afsS promoter and, therefore, compete in the integration of signals of phosphate starvation and S-adenosylmethionine (SAM) concentrations. The cross-talk between phosphate control of metabolism, mediated by the PhoR–PhoP system, and the pleiotropic orphan nitrogen regulator GlnR, is very interesting; PhoP represses GlnR and other nitrogen metabolism genes. The mechanisms of control by GlnR of several promoters of ATP binding cassettes (ABC) sugar transporters and carbon metabolism are highly elaborated. Another important cross-talk that governs nitrogen metabolism involves the competition between GlnR and the transcriptional factor MtrA. GlnR and MtrA exert opposite effects on expression of nitrogen metabolism genes. MtrA, under nitrogen rich conditions, represses expression of nitrogen assimilation and regulatory genes, including GlnR, and competes with GlnR for the GlnR binding sites. Strikingly, these sites also bind to PhoP. Novel examples of interacting transcriptional factors, discovered recently, are discussed to provide a broad view of this interactions. Altogether, these findings indicate that cross-talks between the major transcriptional factors protect the cell metabolic balance. A detailed analysis of the transcriptional factors binding sequences suggests that the transcriptional factors interact with specific regions, either by overlapping the recognition sequence of other factors or by binding to adjacent sites in those regions. Additional interactions on the regulatory backbone are provided by sigma factors, highly phosphorylated nucleotides, cyclic dinucleotides, and small ligands that interact with cognate receptor proteins and with TetR-type transcriptional regulators. We propose to define the signal integration DNA regions (so called integrator sites) that assemble responses to different stress, nutritional or environmental signals. These integrator sites constitute nodes recognized by two, three, or more transcriptional factors to compensate the unbalances produced by metabolic stresses. This interplay mechanism acts as a safety net to prevent major damage to the metabolism under extreme nutritional and environmental conditions.

Mutational landscape screening of methylene tetrahydrofolate reductase to predict homocystinuria associated variants: An integrative computational approach

Methylene tetrahydrofolate reductase (MTHFR) is a flavoprotein, involved in one-carbon pathway and is responsible for folate and homocysteine metabolism. Regulation of MTHFR is pivotal for maintaining the cellular concentrations of methionine and SAM (S-adenosyl methionine) which are essential for the synthesis of nucleotides and amino acids, respectively. Therefore, mutations in MTHFR leads to its dysfunction resulting in conditions like homocystinuria, cardiovascular diseases, and neural tube defects in infants. Among these conditions, homocystinuria has been highly explored, as it manifests ocular disorders, cognitive disorders and skeletal abnormalities. Hence, in this study, we intend to explore the mutational landscape of human MTHFR isoform-1 (h.MTHFR-1) to decipher the most pathogenic variants pertaining to homocystinuria. Thus, a multilevel stringent prioritization of non-synonymous mutations in h.MTHFR-1 by integrative machine learning approaches was implemented to delineate highly deleterious variants based on its pathogenicity, impact on structural stability and functionality. Subsequently, extended molecular dynamics simulations and molecular docking studies were also integrated in order to prioritize the mutations that perturbs structural stability and functionality of h.MTHFR-1. In addition, displacement of Loop (Arg157-Tyr174) and helix α9 (His263-Ser272) involved in open/closed conformation of substrate binding domain were also probed to confirm the functional loss. On juxtaposed analysis, it was inferred that among 126 missense mutations screened, along with known pathogenic mutations (H127 T, A222 V, T227 M, F257 V and G387D) predicted that W500C, P254S and D585 N variants could be potentially driving homocystinuria. Thus, uncovering the prospects for inclusion of these mutations in diagnostic panels based on further experimental validations.

Unraveling the role of quorum sensing-dependent metabolic homeostasis of the activated methyl cycle in a cooperative population of Burkholderia glumae

The activated methyl cycle (AMC) is responsible for the generation of S-adenosylmethionine (SAM), which is a substrate of N-acylhomoserine lactone (AHL) synthases. However, it is unknown whether AHL-mediated quorum sensing (QS) plays a role in the metabolic flux of the AMC to ensure cell density-dependent biosynthesis of AHL in cooperative populations. Here we show that QS controls metabolic homeostasis of the AMC critical for AHL biosynthesis and cellular methylation in Burkholderia glumae, the causal agent of rice panicle blight. Activation of genes encoding SAM-dependent methyltransferases, S-adenosylhomocysteine (SAH) hydrolase, and methionine synthases involved in the AMC by QS is essential for maintaining the optimal concentrations of methionine, SAM, and SAH required for bacterial cooperativity as cell density increases. Thus, the absence of QS perturbed metabolic homeostasis of the AMC and caused pleiotropic phenotypes in B. glumae. A null mutation in the SAH hydrolase gene negatively affected AHL and ATP biosynthesis and the activity of SAM-dependent methyltransferases including ToxA, which is responsible for the biosynthesis of a key virulence factor toxoflavin in B. glumae. These results indicate that QS controls metabolic flux of the AMC to secure the biosynthesis of AHL and cellular methylation in a cooperative population.

Effects of Proanthocyanidins on Arsenic Methylation Metabolism and Efflux in Human Hepatocytes L-02.

This study investigated the effects of proanthocyanidins (PC) on arsenic methylation metabolism and efflux in human hepatocytes (L-02), as well as the relationships between PC and GSH, MRP1 and other molecules. Cells were randomly divided into blank control group, arsenic trioxide exposure group (ATO, As2O3, 25μmol/L), and PC-treated arsenic exposure group (10, 25, 50mg/L). After 24/48h, the contents of different forms of arsenic were determined, and the methylation indexes were calculated. Intracellular S-adenosyl methionine (SAM), arsenic (+3 oxidation state) methyltransferase (AS3MT), multidrug resistance-associated protein 1 (MRP1), and reduced glutathione (GSH) were ascertained. Changing trends were observed and the correlation between arsenic metabolism and efflux related factors and arsenic metabolites was analyzed. We observed that cells showed increased levels of content/constituent ratio of methyl arsenic, primary/secondary methylation index, methylation growth efficiency/rate, and the difference of methyl arsenic content in cells and culture medium (P<0.05, resp.). Compared with ATO exposure group, the intracellular SAM content in PC-treated group decreased, and the contents of GSH, AS3MT, and MRP1 increased (P<0.05, resp.). There was a positive correlation between the content of intracellular GSH/AS3MT and methyl arsenic. The content of MRP1 was positively correlated with the difference of methyl arsenic content in cell and culture medium; conversely, the SAM content was negatively correlated with intracellular methyl arsenic content (P<0.05, resp.). Taken together, these results prove that PC can promote arsenic methylation metabolism and efflux in L-02 cells, which may be related to the upregulation of GSH, MRP1, and AS3MT levels by PC.

S-adenosylhomocysteine as a methyl transfer catalyst in biocatalytic methylation reactions

S-adenosylmethionine-dependent methyltransferases form a large family of enzymes that can catalyse regio-, chemo- and stereospecific methylation of complex natural products. These enzymes could be very useful tools for the chemoenzymatic production and diversification of natural or artificial compounds. Despite this potential, in vitro applications of methyltransferases are limited by their requirement for S-adenosylmethionine as a stoichiometric methyl donor. The chemical complexity, instability, high cost and poor atom economy of this reagent prevent preparative in vitro methylation reactions from becoming routine protocols in natural product research and viable options for process development. In this Article we demonstrate that C-, N- and O-specific methyltransferases can be combined with halide methyltransferases to form enzyme cascades that require only catalytic concentrations of S-adenosylmethionine and use methyl iodide as the stoichiometric methyl donor. Regenerating expensive S-adenosylmethionine (SAM) in enzymatic in vitro reactions is challenging—but important for the commercial scope of SAM-dependent enzymes. This work reports a simple two-enzyme cascade for the in vitro regeneration of SAM for the enzymatic methylation of diverse substrates.

Semi-rationally engineered variants of S-adenosylmethionine synthetase from Escherichia coli with reduced product inhibition and improved catalytic activity

S-adenosylmethionine synthetase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM) from ATP and L-methionine. SAM is the major methyl donor for more than 100 transmethylation reactions. It is also a common cosubstrate involved in transsulfuration and aminopropylation. However, product inhibition largely restrains the activity of MAT and limits the enzymatic synthesis of SAM. In this research, the product inhibition of MAT from Escherichia coli was reduced via semi-rational modification. A triple variant (Variant III, I303 V/I65 V/L186 V) showed a 42-fold increase in Ki,ATP and a 2.08-fold increase in specific activity when compared to wild-type MAT. Its Ki,ATP was 0.42 mM and specific acitivity was 3.78 ± 0.19 U/mg. Increased Ki,ATP means reduced product inhibition which enhances SAM accumulation. The SAM produced by Variant III could reach to 3.27 mM while SAM produced by wild-type MAT was 1.62 mM in the presence of 10 mM substrates. When the residue in 104th of Variant III was further optimized by site-saturated mutagenesis, the specific activity of Variant IV (I303 V/I65 V/L186 V/N104 K) reached to 6.02 ± 0.22 U/mg at 37 °C, though the SAM concentration decreased to 2.68 mM with 10 mM substrates. Analysis of protein 3D structure suggests that changes in hydrogen bonds or other ligand interactions around active site may account for the variety of product inhibition and enzyme activity. The Variant III and Variant IV with reduced inhibition and improved enzyme activity in the study would be more suitable candidates for SAM production in the future.

TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAM.

BackgroundHepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown.MethodsWe performed pharmacoproteomic profiling of the mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. The target gene MAT2A was overexpressed or knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional activity on MAT2A promoter via luciferase assay. Intracellular SAM contents were analyzed by LC-MS method.FindingsWe found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice, and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65, i.e., activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393.InterpretationThis study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis.FundThis work was supported by National Natural Science Foundation of China (No. 81500469, 81573873, 81774196 and 31800693), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502).

Association of neural tube defects with maternal alterations and genetic polymorphisms in one-carbon metabolic pathway

BackgroundNeural tube defects (NTDs) are birth defects of the brain, spine, or spinal cord invoked by the insufficient intake of folic acid in the early stages of pregnancy and have a complex etiology involving both genetic and environmental factors. So the study aimed to explore the association between alterations in maternal one-carbon metabolism and NTDs in the offspring.MethodsWe conducted a case-control study to get a deeper insight into this association, as well as into the role of genetic polymorphisms. Plasma concentrations of folate, homocysteine (Hcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and genotypes and alleles distributions of 52 SNPs in 8 genes were compared for 61 women with NTDs-affected offspring and 61 women with healthy ones.ResultsThere were significant differences between groups with regard to plasma folate, SAM, SAH and SAM/SAH levels. Logistic regression results revealed a significant association between maternal plasma folate level and risk of NTDs in the offspring. For MTHFD1 rs2236225 polymorphism, mothers having GA genotype and A allele exhibited an increased risk of NTDs in the offspring (OR = 2.600, 95%CI: 1.227–5.529; OR = 1.847, 95%CI: 1.047–3.259). For MTHFR rs1801133 polymorphism, mothers having TT and CT genotypes were more likely to affect NTDs in the offspring (OR = 4.105, 95%CI: 1.271–13.258; OR = 3.333, 95%CI: 1.068–10.400). Moreover, mothers carrying T allele had a higher risk of NTDs in the offspring (OR = 1.798, 95%CI: 1.070–3.021). For MTRR rs1801394 polymorphism, the frequency of G allele was significantly higher in cases than in controls (OR = 1.763, 95%CI: 1.023–3.036). Mothers with NTDs-affected children had higher AG genotype in RFC1 rs1051226 polymorphism than controls, manifesting an increased risk for NTDs (OR = 3.923, 95%CI: 1.361–11.308).ConclusionFolic acid deficiency, MTHFD1 rs2236225, MTHFR rs1801133, MTRR rs1801349 and RFC1 rs1051226 polymorphisms may be maternal risk factors of NTDs.

Depletion of S-adenosylmethionine impacts on ribosome biogenesis through hypomodification of a single rRNA methylation

S-adenosylmethionine (SAM) is an essential metabolite and a methyl group donor in all living organisms. The intracellular SAM concentration is tightly regulated, and depletion causes hypomethylation of substrates, growth defects and pathological consequences. In the emerging field of epitranscriptomics, SAM-dependent RNA methylations play a critical role in gene expression. Herein, we analyzed the methylation status of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) in Escherichia coli Δmtn strain in which cellular SAM was down-regulated, and found hypomodification of several methylation sites, including 2′-O-methylation at position 2552 (Um2552) of 23S rRNA. We observed severe growth defect of the Δmtn strain with significant accumulation of 45S ribosomal precursor harboring 23S rRNA with hypomodified Um2552. Strikingly, the growth defect was partially restored by overexpression of rlmE encoding the SAM-dependent methyltransferase responsible for Um2552. Although SAM is involved not only in rRNA methylation but also in various cellular processes, effects on ribosome biogenesis contribute substantially to the observed defects on cell proliferation.

TGF-β1/P65/MAT2A Pathway Regulates Liver Fibrogenesis via Intracellular SAM

Background: Hepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown. Methods: We performed proteomic profiling of mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. Our target Mat2a was overexpressed and knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional and MAT2A promoter activation via luciferase assay. Intracellular SAM contents were analyzed by LC/MS method. Findings: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice model and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65 and activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. Interpretation: This study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. Funding: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873 and 81774196), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The animal experiment was approved by Science and Technology Commission of Shanghai Municipality, and all experimental procedures were performed according to the ethical guidelines of Animal care and use committee, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Behavioral and metabolic effects of S-adenosylmethionine and imipramine in the Flinders Sensitive Line rat model of depression.

Depression is associated with dysregulation of methyl group metabolism such as low S-adenosylmethionine (SAM). We previously reported that Flinders Sensitive Line (FSL) rats, an animal model of depression, had lower concentrations of liver SAM than the control rats, Flinders Resistant Line (FRL) rats. The present study investigated if SAM supplementation may correct liver SAM and behavioral abnormalities in this model. Moreover, we compared one-carbon (C1) metabolites, neurotransmitters, and gastrointestinal (GI) transit in SAM-treated versus imipramine (IMI)-treated animals. FSL rats received vehicle, IMI, SAM, or IMI + SAM (n = 9-10 per group) once daily through oral gavage for 4 weeks; FRL rats received vehicle. Behavior was assessed using standard tests for locomotion, cognition, and depressive-like behavior. Monoamine neurotransmitters and C1 metabolites were measured using UHPLC-ECD and UPLC-MS/MS, respectively. Compared to FRL rats, FSLs had lower liver SAM, higher plasma serotonin, lower hippocampal dopamine and serotonin turnover, and faster GI transit. Behaviorally, FSL rats showed impaired cognitive performance as well as increased depressive-like behavior compared to FRLs. Coadministration of IMI and SAM seemed to have adverse effects on spatial memory. SAM or IMI administration did not reverse C1 metabolites, neurotransmitters, or GI transit in FSLs. Despite low liver SAM in FSL rats, orally administered SAM did not show antidepressant effects in this specific animal model of depression.

Hepatic accumulation of S-adenosylmethionine in hamsters with non-alcoholic fatty liver disease associated with metabolic syndrome under selenium and vitamin E deficiency.

Progression of non-alcoholic fatty liver disease (NAFLD) in the context of metabolic syndrome (MetS) is only partially explored due to the lack of preclinical models. In order to study the alterations in hepatic metabolism that accompany this condition, we developed a model of MetS accompanied by the onset of steatohepatitis (NASH) by challenging golden hamsters with a high-fat diet low in vitamin E and selenium (HFD), since combined deficiency results in hepatic necroinflammation in rodents. Metabolomics and transcriptomics integrated analyses of livers revealed an unexpected accumulation of hepatic S-Adenosylmethionine (SAM) when compared with healthy livers likely due to diminished methylation reactions and repression of GNMT. SAM plays a key role in the maintenance of cellular homeostasis and cell cycle control. In agreement, analysis of over-represented transcription factors revealed a central role of c-myc and c-Jun pathways accompanied by negative correlations between SAM concentration, MYC expression and AMPK phosphorylation. These findings point to a drift of cell cycle control toward senescence in livers of HFD animals, which could explain the onset of NASH in this model. In contrast, hamsters with NAFLD induced by a conventional high-fat diet did not show SAM accumulation, suggesting a key role of selenium and vitamin E in SAM homeostasis. In conclusion, our results suggest that progression of NAFLD in the context of MetS can take place even in a situation of hepatic SAM excess and that selenium and vitamin E status might be considered in current therapies against NASH based on SAM supplementation.

The Antisteatotic and Hypolipidemic Effect of Insect Meal in Obese Zucker Rats is Accompanied by Profound Changes in Hepatic Phospholipid and 1-Carbon Metabolism.

The hypothesis is tested that insect meal, which has a low methionine content, reduces the hepatic phosphatidylcholine (PC):phosphatidylethanolamine (PE) ratio, which is a critical determinant of hepatic lipid synthesis, by decreasing availability of the methionine metabolite S-adenosylmethionine (SAM). Obese rats (n = 24) are randomly divided into two groups (Obese Casein and Obese Insect) of 12 rats each. In addition, lean rats (n = 12) are used as control group (LC). Groups LC and OC receive a control diet with casein as protein source, whereas in the OI group, casein is replaced isonitrogenously by insect meal, which is found to be less digestible (-12% units). Plasma and liver concentrations of lipids and hepatic expression of lipid synthesizing genes are reduced in the OI group compared to the OC group. Plasma and liver concentration of PC and the PC:PE ratio are decreased in the OI group compared to the OC group, while hepatic concentration of SAM and the hepatic SAM:S-adenosylhomocysteine (SAH) ratio is lower in the OI group than in the OC group. The decrease of the hepatic PC:PE ratio is probably a key mechanism explaining the pronounced antisteatotic and hypolipidemic action of insect meal in obese rats.

The structure of the SAM/SAH-binding riboswitch.

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)—the product of SAM-dependent methyl group transfer reactions—and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational ‘OFF’-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.

Tunicamycin-Induced ER Stress is Accompanied with Oxidative Stress via Abrogation of Sulfur Amino Acids Metabolism in the Liver.

Endoplasmic reticulum (ER) stress is involved in non-alcoholic fatty liver disease (NAFLD), but the relationship between oxidative stress, another well-known risk factor of NAFLD, and ER stress has yet to be elucidated. In this study, we treated mice with tunicamycin (TM) (2 mg/kg body weight) for 48 h to induce ER stress in the liver and examined the metabolic pathway that synthesizes the endogenous antioxidant, glutathione (GSH). Tunicamycin (TM) treatment significantly increased mRNA levels of CHOP and GRP78, and induced lipid accumulation in the liver. Lipid peroxidation in the liver tissue also increased from TM treatment (CON vs. TM; 3.0 ± 1.8 vs. 11.1 ± 0.8 nmol MDA/g liver, p < 0.001), which reflects an imbalance between the generation of reactive substances and antioxidant capacity. To examine the involvement of GSH synthetic pathway, we determined the metabolomic changes of sulfur amino acids in the liver. TM significantly decreased hepatic S-adenosylmethionine concentration in the methionine cycle. The levels of cysteine in the liver were increased, while taurine concentration was maintained and GSH levels profoundly decreased (CON vs. TM; 8.7 ± 1.5 vs. 5.4 ± 0.9 µmol GSH/g liver, p < 0.001). These results suggest that abnormal cysteine metabolism by TM treatment resulted in a decrease in GSH, followed by an increase in oxidative stress in the liver. In HepG2 cells, decreased GSH levels were examined by TM treatment in a dose dependent manner. Furthermore, pretreatment with TM in HepG2 cells potentiated oxidative cell death, by exacerbating the effects of tert-butyl hydroperoxide. In conclusion, TM-induced ER stress was accompanied by oxidative stress by reducing the GSH synthesis, which made the liver more susceptible to oxidative stress.

Disruption of Brain Redox Homeostasis, Microglia Activation and Neuronal Damage Induced by Intracerebroventricular Administration of S-Adenosylmethionine to Developing Rats.

S-Adenosylmethionine (AdoMet) concentrations are highly elevated in tissues and biological fluids of patients affected by S-adenosylhomocysteine hydrolase deficiency. This disorder is clinically characterized by severe neurological symptoms, whose pathophysiology is not yet established. Therefore, we investigated the effects of intracerebroventricular administration of AdoMet on redox homeostasis, microglia activation, synaptophysin levels, and TAU phosphorylation in cerebral cortex and striatum of young rats. AdoMet provoked significant lipid and protein oxidation, decreased glutathione concentrations, and altered the activity of important antioxidant enzymes in cerebral cortex and striatum. AdoMet also increased reactive oxygen (2',7'-dichlorofluorescein oxidation increase) and nitrogen (nitrate and nitrite levels increase) species generation in cerebral cortex. Furthermore, the antioxidants N-acetylcysteine and melatonin prevented most of AdoMet-induced pro-oxidant effects in both cerebral structures. Finally, we verified that AdoMet produced microglia activation by increasing Iba1 staining and TAU phosphorylation, as well as reduced synaptophysin levels in cerebral cortex. Taken together, it is presumed that impairment of redox homeostasis possibly associated with microglia activation and neuronal dysfunction caused by AdoMet may represent deleterious pathomechanisms involved in the pathophysiology of brain damage in S-adenosylhomocysteine hydrolase deficiency.

Caloric restriction can affect one-carbon metabolism during pregnancy in the rat: A transgenerational model

One-carbon metabolism is critical to pregnancy outcomes, because it determines the availability of nutrients involved in cell divisions and DNA methylation. The aim of this study was to analyze how 50% prenatal calorie restriction affected one-carbon metabolism in pregnant Wistar rats of the F0 to F2 generations. Mean choline (p < 0.001), betaine (p < 0.001), and S-adenosylmethionine (SAM) (p < 0.05) concentrations were respectively about 40%, 45%, and 20% lower in the F0_R (R – restricted diet) than in the F0_C (C – control diet). Homocysteine, S-adenosylhomocysteine (SAH), and trimethylamine oxide concentrations were unaffected. In the F1_R, the SAM-to-SAH ratio was 25% higher (p < 0.05) than in the F1_C. No differences between the C and R groups were observed in the F2 generation. The SAM concentrations in the F1_R were higher than in the F0_R and the F2_R (p < 0.01). The relative transcript levels of Mat1a, Bhmt, Cbs, Pemt, and Mthfr were only slightly affected by the diet, with changes of less than a factor of 2.0. Cbs activity in the F2_R was significantly higher than in the F2_C (p < 0.001). Food deprivation may affect one-carbon metabolism in pregnant rats, but it does not stimulate persistent metabolic changes that can be observed during the pregnancy of their progeny of the F1 or F2 generations.

Methylenetetrahydrofolate reductase modulates methyl metabolism and lignin monomer methylation in maize.

The brown midrib2 (bm2) mutant of maize, which has a modified lignin composition, contains a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. Here, we show that a MITE transposon insertion caused down-regulation of MTHFR, with an accompanying decrease in 5-methyl-tetrahydrofolate and an increase in 5, 10-methylene-tetrahydrofolate and tetrahydrofolate in the bm2 mutant. Furthermore, MTHFR mutation did not change the content of S-adenosyl methionine (SAM), the methyl group donor involved in the biosynthesis of guaiacyl and syringyl lignins, but increased the level of S-adenosyl homocysteine (SAH), the demethylation product of SAM. Moreover, competitive inhibition of the maize caffeoyl CoA O-methyltransferase (CCoAOMT) and caffeic acid O-methyltransferase (COMT) enzyme activities by SAH was found, suggesting that the SAH/SAM ratio, rather than the concentration of SAM, regulates the transmethylation reactions of lignin intermediates. Phenolic profiling revealed that caffeoyl alcohol glucose derivatives accumulated in the bm2 mutant, indicating impaired 3-O-methylation of monolignols. A remarkable increase in the unusual catechyl lignin in the mutant demonstrates that MTHFR down-regulation mainly affects guaiacyl lignin biosynthesis, consistent with the observation that CCoAOMT is more sensitive to SAH inhibition than COMT. This study uncovered a novel regulatory mechanism in lignin biosynthesis, which may offer an effective approach to utilizing lignocellulosic feedstocks in the future.

Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis.

Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert L-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as ΔmetK2. Following a reduction of the intracellular SAM concentration, ΔmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X.