Methionine adenosyltransferase deficiency (MATD) is a rare metabolic disorder caused by mono- or biallelic MAT1A mutations that are not yet well understood. Of the 4,065,644 neonates screened between November 2010 and December 2021, 35 individuals have been diagnosed with an estimated incidence of 1: 116,161 by a cutoff value of methionine 82.7 μmol/L and follow-up over 11 years. MATD patients with autosomal recessive (AR) type had higher clinical and genetic heterogeneity than those with autosomal dominant (AD) type. Fifteen unrelated AD patients harbored one well-known dominant variant, c.791 G>A or c.776 C>T, and were clinically unaffected with a mean plasma methionine (Met) value <300 μmol/L. Twenty AR cases have unique genotypes and presented a wide range of clinical abnormalities from asymptomatic to white matter lesions. Of them, 10 AR patients displayed severe manifestations, such as verbal difficulty, motor delay, development delay, and white matter lesions, with mean Met >500 μmol/L and thereby were treated with a methionine-restricted diet alone or in combination with betaine, folate, or vitamin B6, and were healthy finally. Neurological abnormalities were evidenced in two patients (P16 and P27) with Met values >800 μmol/L by MRI scan. Neurological abnormalities were reversed here by liver transplantation or by the determination of S-adenosylmethionine supplementation. Additionally, 38 variants of MAT1A were distributed within patients and carriers, of which 24 were novel and mostly predicted to be damaged. Our findings with an extensive clinical and genetic dataset provided new insights into its diagnosis and treatment and will be helpful for its optimal management in the future.

Enhancer of zeste homologue 2 (EZH2) is the enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2), which plays an important role in post-translational modifications of histones. In this study, we designed and synthesized a new series EZH2 covalent inhibitors that have rarely been reported. Biochemical studies and mass spectrometry provide information that SKLB-03220 could covalently bind to the S-adenosylmethionine (SAM) pocket of EZH2. Besides, SKLB-03220 was highly potent for EZH2MUT, while exhibiting weak activities against other tested histone methyltransferases (HMTs) and kinases. Moreover, SKLB-03220 displayed noteworthy potency against ovarian cancer cell lines and continuously abolished H3K27me3 after washing out. Furthermore, oral administration of SKLB-03220 significantly inhibited tumor growth in PA-1 xenograft model without obvious adverse effects. Taken together, SKLB-03220 is a potent, selective EZH2 covalent inhibitor with noteworthy anticancer efficacy both in vitro and in vivo.

Due to the emergence of multidrug resistant pathogens, it is imperative to identify new targets for antibiotic drug discovery. The S-adenosylhomocysteine (SAH) nucleosidase enzyme is a promising target for antimicrobial drug development due to its critical functions in multiple bacterial processes including recycling of toxic byproducts of S-adenosylmethionine (SAM)-mediated reactions and producing the precursor of the universal quorum sensing signal, autoinducer-2 (AI-2). Riboswitches are structured RNA elements typically used by bacteria to precisely monitor and respond to changes in essential bacterial processes, including metabolism. Natural riboswitches fused to a reporter gene can be exploited to detect changes in metabolism or in physiological signaling. We performed a high-throughput screen (HTS) using an SAH-riboswitch controlled β-galactosidase reporter gene in Escherichia coli to discover small molecules that inhibit SAH recycling. We demonstrate that the assay strategy using SAH riboswitches to detect the effects of SAH nucleosidase inhibitors can quickly identify compounds that penetrate the barriers of Gram-negative bacterial cells and perturb pathways involving SAH.

Mixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host's metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth.

Breastfeeding is the gold standard for early nutrition. Metabolites from the one-carbon metabolism pool are crucial for infant development. The aim of this study is to compare the breast-milk one-carbon metabolic profile to other biofluids where these metabolites are present, including cord and adult blood plasma as well as cerebrospinal fluid. Breast milk (n = 142), cord blood plasma (n = 23), maternal plasma (n = 28), aging adult plasma (n = 91), cerebrospinal fluid (n = 92), and infant milk formula (n = 11) samples were analyzed by LC-MS/MS to quantify choline, betaine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, total homocysteine, and cystathionine. Differences between groups were visualized by principal component analysis and analyzed by Kruskal-Wallis test. Correlation analysis was performed between one-carbon metabolites in human breast milk. Principal component analysis based on these metabolites separated breast milk samples from other biofluids. The S-adenosylmethionine (SAM) concentration was significantly higher in breast milk compared to the other biofluids and was absent in infant milk formulas. Despite many significant correlations between metabolites in one-carbon metabolism, there were no significant correlations between SAM and methionine or total homocysteine. Together, our data indicate a high concentration of SAM in breast milk, which may suggest a strong demand for this metabolite during infant early growth while its absence in infant milk formulas may indicate the inadequacy of this vital metabolic nutrient.

SARS-CoV-2 nsp14 guanine-N7-methyltransferase plays an important role in the viral RNA translation process by catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to viral mRNA cap. We report a structure-guided design and synthesis of 3-(adenosylthio)benzoic acid derivatives as nsp14 methyltransferase inhibitors resulting in compound 5p with subnanomolar inhibitory activity and improved cell membrane permeability in comparison with the parent inhibitor. Compound 5p acts as a bisubstrate inhibitor targeting both SAM and mRNA-binding pockets of nsp14. While the selectivity of 3-(adenosylthio)benzoic acid derivatives against human glycine N-methyltransferase was not improved, the discovery of phenyl-substituted analogs 5p,t may contribute to further development of SARS-CoV-2 nsp14 bisubstrate inhibitors.

Physcion is one of natural anthraquinones, registered as a novel plant-derived fungicide due to its excellent prevention of plant disease. However, the current production of physcion via plant extraction limits its yield promotion and application. Here, a pair of polyketide synthases (PKS) in emodin biosynthesis were used as probes to mining the potential O-methyltransferase (OMT) responsible for physcion biosynthesis. Further refinement using the phylogenetic analysis of the mined OMTs revealed a distinct OMT (AcOMT) with the ability of transferring a methyl group to C-6 hydroxyl of emodin to form physcion. Through introducing AcOMT, we successfully obtained the de novo production of physcion in Aspergillus nidulans. The physcion biosynthetic pathway was further rationally engineered by expressing the decarboxylase genes from different fungi. Finally, the titer of physcion reached to 64.6 mg/L in shake-flask fermentation through enhancing S-adenosylmethionine supply. Our work provides a native O-methyltransferase for physcion biosynthesis and lays the foundation for further improving the production of physcion via a sustainable route. KEY POINTS: • Genome mining of the native O-methyltransferase responsible for physcion biosynthesis • De novo biosynthesis of physcion in the engineered Aspergillus nidulans • Providing an alternative way to produce plant-derived fungicide physcion.

Colorectal cancer (CRC) is a common gastrointestinal malignancy with high morbidity and fatality. Chemotherapy, as traditional therapy for CRC, has exerted well antitumor effect and greatly improved the survival of CRC patients. Nevertheless, chemoresistance is one of the major problems during chemotherapy for CRC and significantly limits the efficacy of the treatment and influences the prognosis of patients. To overcome chemoresistance in CRC, many strategies are being investigated. Here, we review the common and novel measures to combat the resistance, including drug repurposing (nonsteroidal anti-inflammatory drugs, metformin, dichloroacetate, enalapril, ivermectin, bazedoxifene, melatonin, and S-adenosylmethionine), gene therapy (ribozymes, RNAi, CRISPR/Cas9, epigenetic therapy, antisense oligonucleotides, and noncoding RNAs), protein inhibitor (EFGR inhibitor, S1PR2 inhibitor, and DNA methyltransferase inhibitor), natural herbal compounds (polyphenols, terpenoids, quinones, alkaloids, and sterols), new drug delivery system (nanocarriers, liposomes, exosomes, and hydrogels), and combination therapy. These common or novel strategies for the reversal of chemoresistance promise to improve the treatment of CRC.

Despite marked success in treatment with immune checkpoint inhibitor (CPI), only a third of patients are responsive. Thus, melanoma still has one of the highest prevalence and mortality rates; which has led to a search for novel combination therapies that might complement CPI. Aberrant methylomes are one of the mechanisms of resistance to CPI therapy. S-adenosylmethionine (SAM), methyl donor of important epigenetic processes, has significant anti-cancer effects in several malignancies; however, SAM's effect has never been extensively investigated in melanoma. We demonstrate that SAM modulates phenotype switching of melanoma cells and directs the cells towards differentiation indicated by increased melanogenesis (melanin and melanosome synthesis), melanocyte-like morphology, elevated Mitf and Mitf activators' expression, increased antigen expression, reduced proliferation, and reduced stemness genes' expression. Consistently, providing SAM orally, reduced tumor growth and progression, and metastasis of syngeneic BRAF mutant and wild-type (WT) melanoma mouse models. Of note, SAM and anti-PD-1 antibody combination treatment had enhanced anti-cancer efficacy compared to monotherapies, showed significant reduction in tumor growth and progression, and increased survival. Furthermore, SAM and anti-PD-1 antibody combination triggered significantly higher immune cell infiltration, higher CD8+ T cells infiltration and effector functions, and polyfunctionality of CD8+ T cells in YUMMER1.7 tumors. Therefore, SAM combined with CPI provides a novel therapeutic strategy against BRAF mutant and WT melanomas and provides potential to be translated into clinic.

Oxidative stress and neuroinflammation play crucial roles in aging. S-adenosylmethionine (SAM), a popular supplement, is a potential antioxidant and candidate therapy for depression. This study aimed to evaluate the neuroprotective effects of SAM on D-galactose-induced brain aging and explore its underlying mechanisms. Brain aging model was established with D-galactose (180 mg/kg/day) for 8 weeks. During the last 4 weeks, SAM (16 mg/kg) was co-administrated with D-galactose. Behavior tests were used to assess cognitive function and depression-like behaviors of rats. Results showed that cognitive impairment and depression-like behaviors were reversed by SAM. SAM reduced neuronal cell loss, increased brain-derived neurotrophic factor level in the hippocampus, inhibited amyloid-β level and microglia activation, as well as pro-inflammatory factors levels in the hippocampus and serum. Further, SAM enhanced antioxidant capacity and attenuated cholinergic damage by reducing malondialdehyde levels, increasing acetylcholine levels, expression levels of α7 nicotinic acetylcholine receptor (α7nAChR), nuclear factor erythrocyte 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the hippocampus. Above all, SAM has a potential neuroprotective effect on ameliorating cognitive impairment in brain aging, which is related to inhibition of oxidative stress and neuroinflammation, as well as α7nAChR signals. DATA AVAILABILITY: Data will be made available on request.

The cholesterol-oxidized metabolite 27-hydroxycholesterol (27-OHC) is synthesized by CYP27A1, which is a key factor in vitamin D and oxysterol metabolism. Both vitamin D and 27-OHC are considered to play important roles in Alzheimer's disease (AD). The study aims to research the effects of co-supplementation of vitamin D, folic acid, and vitamin B12 on learning and memory ability in vitamin D-deficient mice, and to explore the underlying mechanism. In this study, C57BL/6J mice were fed a vitamin D-deficient diet for 13 weeks to establish a vitamin D-deficient mice model. The vitamin D-deficient mice were then orally gavaged with vitamin D (VD), folic acid (FA), and vitamin B12 (VB12) alone or together for eight weeks. Following the gavage, the learning and memory ability of the mice were evaluated by Morris Water Maze and Novel object recognition test. The CYP27A1-related gene and protein expressions in the liver and brain were determined by qRT-PCR. The serum level of 27-OHC was detected by HPLC-MS. Serum levels of 25(OH)D, homocysteine (Hcy), and S-Adenosylmethionine (SAM) were measured by ELISA. After feeding with the vitamin D-deficient diet, the mice performed longer latency to a platform (p < 0.001), lower average speed (p = 0.026) in the Morris Water Maze, a lower time discrimination index (p = 0.009) in Novel object recognition, and performances were reversed after vitamin D, folic acid and vitamin B12 supplementation alone or together (p < 0.05). The gene expressions of CYP27A1 in the liver and brain were upregulated in the vitamin D-deficiency (VDD) group compared with the control (CON) group (p = 0.015), while it was downregulated in VDD + VD and VDD + VD-FA/VB12 groups compared with the VDD group (p < 0.05), with a similar trend in the protein expression of CYP27A1. The serum levels of 27-OHC were higher in the VDD group, compared with CON, VDD + VD, and VDD + VD-FA/VB12 group (p < 0.05), and a similar trend was found in the brain. The serum 25(OH)D levels were significantly decreased in the vitamin D-deficiency group (p = 0.008), and increased in the vitamin D-supplemented group (p < 0.001). The serum levels of SAM were higher in the B vitamins-supplemented group, compared with CON and VDD groups (p < 0.05). This study suggests that CYP27A1 expression may be involved in the mechanism of learning and memory impairment induced by vitamin D deficiency. Co-supplementation with vitamin D, folic acid, and vitamin B12 significantly reverses this effect by affecting the expression of CYP27A1, which in turn regulates the metabolism of 27-OHC, 25(OH)D, and SAM.

In plants, methylation is a common step in specialized metabolic pathways, leading to a vast diversity of natural products. The methylation of these small molecules is catalyzed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which are categorized based on the methyl-accepting atom (O, N, C, S, or Se). These methyltransferases are responsible for the transformation of metabolites involved in plant defense response, pigments, and cell signaling. Plant natural product methyltransferases are part of the Class I methyltransferase-superfamily containing the canonical Rossmann fold. Recent advances in genomics have accelerated the functional characterization of plant natural product methyltransferases, allowing for the determination of substrate specificities and regioselectivity and further realizing the potential for enzyme engineering. This review compiles known biochemically characterized plant natural product methyltransferases that have contributed to our knowledge in the diversification of small molecules mediated by methylation steps.

Ladderane lipids (found in the membranes of anaerobic ammonium-oxidizing [anammox] bacteria) have unique ladder-like hydrophobic groups, and their highly strained exotic structure has attracted the attention of scientists. Although enzymes encoded in type II fatty acid biosynthesis (FASII) gene clusters in anammox bacteria, such as S-adenosyl-l-methionine (SAM)-dependent enzymes, have been proposed to construct a ladder-like structure using a substrate connected to acyl carrier protein from anammox bacteria (AmxACP), no experimental evidence to support this hypothesis was reported to date. Here, we report the crystal structure of a SAM-dependent methyltransferase from anammox bacteria (AmxMT1) that has a substrate and active site pocket between a class I SAM methyltransferase-like core domain and an additional α-helix inserted into the core domain. Structural comparisons with homologous SAM-dependent C-methyltransferases in polyketide synthase, AmxACP pull-down assays, AmxACP/AmxMT1 complex structure predictions by AlphaFold, and a substrate docking simulation suggested that a small compound connected to AmxACP could be inserted into the pocket of AmxMT1, and then the enzyme transfers a methyl group from SAM to the substrate to produce branched lipids. Although the enzymes responsible for constructing the ladder-like structure remain unknown, our study, for the first time, supports the hypothesis that biosynthetic intermediates connected to AmxACP are processed by SAM-dependent enzymes, which are not typically involved in the FASII system, to produce the ladder-like structure of ladderane lipids in anammox bacteria.

Breast cancer (BCa) is the most prevalent cancer in females and has a high rate of mortality, especially due to increased metastasis to skeletal and non-skeletal sites. Despite the marked clinical accomplishment of immune checkpoint inhibitor (CPI) therapy in patients with several cancers, it has had limited success in luminal subtypes of BCa. Accordingly, recent efforts have focused on combination therapy with CPI, including epigenetic modulators, to increase response rates of CPI in luminal BCa. We have previously shown that S-adenosylmethionine (SAM), the ubiquitous methyl donor, has strong anti-cancer effects in various cancers, including all subtypes of BCa. In the current study, we took a novel approach and examined the effect of CPI alone and in combination with SAM on tumor growth and metastasis in a syngeneic mouse model of luminal B BCa. We showed that SAM decreases cell proliferation, colony-formation (survival), and invasion of luminal B BCa cell lines (Eo771, R221A) in vitro. In in vivo studies, in Eo771 tumor-bearing mice, either SAM or anti-PD-1 antibody treatment alone significantly reduced tumor growth and progression, while the SAM+anti-PD-1 combination treatment had the highest anti-cancer efficacy of all groups. The SAM+anti-PD-1 combination reduced the percentage of animals with lung metastasis, as well as total metastatic lesion area, compared to control. Additionally, the SAM+anti-PD-1 combination significantly reduced the skeletal lesion area and protected tibial integrity to a greater extent than the monotherapies in an Eo771 bone metastasis model. Transcriptome analysis of Eo771 primary tumors revealed significant downregulation of pro-metastatic genes, including Matrix metalloproteinases (MMPs) and related pathways. On the other hand, CD8+ T cell infiltration, CD8+ T cell cytotoxicity (elevated granzymes), and immunostimulatory genes and pathways were significantly upregulated by the combination treatment. The results presented point to a combination of SAM with CPI as a possible treatment for luminal B BCa that should be tested in clinical studies.

Terpenoids constitute the largest class of natural products with complex structures, essential functions, and versatile applications. Creation of new building blocks beyond the conventional five-carbon (C5) units, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate, expands significantly the chemical space of terpenoids. Structure-guided engineering of an S-adenosylmethionine-dependent geranyl diphosphate (GPP) C2-methyltransferase from Streptomyces coelicolor yielded variants converting DMAPP to a new C6 unit, 2-methyl-DMAPP. Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP. Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond. Furthermore, using Escherichia coli as the chassis, we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical trans- and cis-prenyl diphosphates (C5n+1) and terpenoids. This work provides insights into substrate recognition of prenyl diphosphate methyltransferases, and strategies to diversify terpenoids by expanding the building block portfolio.

Specific, regulated modification of RNAs is important for proper gene expression1,2. tRNAs are rich with various chemical modifications that affect their stability and function3,4. 7-Methylguanosine (m7G) at tRNA position 46 is a conserved modification that modulates steady-state tRNA levels to affect cell growth5,6. The METTL1-WDR4 complex generates m7G46 in humans, and dysregulation of METTL1-WDR4 has been linked to brain malformation and multiple cancers7-22. Here we show how METTL1 and WDR4 cooperate to recognize RNA substrates and catalyse methylation. A crystal structure of METTL1-WDR4 and cryo-electron microscopy structures of METTL1-WDR4-tRNA show that the composite protein surface recognizes the tRNA elbow through shape complementarity. The cryo-electron microscopy structures of METTL1-WDR4-tRNA with S-adenosylmethionine or S-adenosylhomocysteine along with METTL1 crystal structures provide additional insights into the catalytic mechanism by revealing the active site in multiple states. The METTL1 N terminus couples cofactor binding with conformational changes in the tRNA, the catalytic loop and the WDR4 C terminus, acting as the switch to activate m7G methylation. Thus, our structural models explain how post-translational modifications of the METTL1 N terminus can regulate methylation. Together, our work elucidates the core and regulatory mechanisms underlying m7G modification by METTL1, providing the framework to understand its contribution to biology and disease.

Neural tube closure disorders, including anencephaly, spina bifida, and encephalocele, cause neural tube defects (NTDs). This congenital disability remained not only a major contributor to the prevalence of stillbirths and neonatal deaths but also a significant cause of lifelong physical disability in surviving infants. NTDs are complex diseases caused by multiple etiologies, levels, and mechanisms. Currently, the pathogenesis of NTDs is considered to be associated with both genetic and environmental factors. Here, we aimed to review the research progress on the etiology and mechanism of NTDs induced by methylation modification caused by folic acid deficiency. Folic acid supplementation in the diet is reported to be beneficial in preventing NTDs. Methylation modification is one of the most important epigenetic modifications crucial for brain neurodevelopment. Disturbances in folic acid metabolism and decreased S-adenosylmethionine levels lead to reduced methyl donors and methylation modification disorders. In this review, we summarized the relationship between NTDs, folic acid metabolism, and related methylation of DNA, imprinted genes, cytoskeletal protein, histone, RNA, and non-coding RNA, so as to clarify the role of folic acid and methylation in NTDs and to better understand the various pathogenesis mechanisms of NTDs and the effective prevention.

Adenosine kinase (ADK) deficiency is a rare inborn error of methionine and adenosine metabolism. So far, a total of 27 patients with ADK deficiency have been reported. Here, we describe the first Polish patient diagnosed with ADK deficiency, aiming to highlight the clinical presentation of disease, emphasize diagnostic difficulties, and report the long-term follow-up. Six-month-old patient presented with cholestatic liver disease, macrocytic anemia, developmental delay, generalized hypotonia, delayed brain myelination, and elevated levels of serum methionine. A decrease of mitochondrial respiratory chain complex II and III activity were found in the postnuclear supernatants obtained from skeletal muscle biopsy. The patient underwent living-donor liver transplantation (LTx) at 14 months of age. Ten-year follow-up after LTx revealed a preserved good liver function, persistent regenerative macrocytic anemia, progressive neurological disease but disappearance of brain MR changes, short stature, and cortisol deficiency. Whole exome sequencing revealed the patient to be affected with two novel ADK variants, which pathogenicity was confirmed biochemically by demonstration of elevated concentration of S-adenosylhomocysteine.

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15-33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g-1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L-1L-met and 0.60 g L-1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L-1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L-1 to 10.1 g L-1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 μM and KD ∼ 0.2 μM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.

Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid, which arises from redox-sensitive methionine metabolism. In plants, Hcy synthesis involves both cystathionine β-lyase and S-adenosylhomocysteine hydrolase activities. Thus, Hcy itself is crucial for de novo methionine synthesis and S-adenosylmethionine recycling, influencing the formation of ethylene, polyamines, and nicotianamine. Research on mammalian cells has shown biotoxicity of this amino acid, as Hcy accumulation triggers oxidative stress and the associated lipid peroxidation process. In addition, the presence of highly reactive groups induces Hcy and Hcy derivatives to modify proteins by changing their structure and function. Currently, Hcy is recognized as a critical, independent hallmark of many degenerative metabolic diseases. Research results indicate that an enhanced Hcy level is also toxic to yeast and bacteria cells. In contrast, in the case of plants the metabolic status of Hcy remains poorly examined and understood. However, the presence of the toxic Hcy metabolites and Hcy over-accumulation during the development of an infectious disease seem to suggest harmful effects of this amino acid also in plant cells. The review highlights potential implications of Hcy metabolism in plant physiological disorders caused by environmental stresses. Moreover, recent research advances emphasize that recognizing the Hcy mode of action in various plant systems facilitates verification of the potential status of Hcy metabolites as bioindicators of metabolism disorders and thus may constitute an element of broadly understood biomonitoring.

The objective of this study was to determine the dose of folate and vitamin B12 in beef heifers fed rumen protected methionine and choline required to maintain increased B12 levels and intermediates of the methionine-folate cycle in circulation. Angus heifers (n = 30; BW = 392.6 ± 12.6 kg) were individually fed and assigned to one of five treatments: 0XNEG: Total mixed ration (TMR) and saline injections at d 0 and 7 of the estrous cycle, 0XPOS: TMR, rumen protected methionine (MET) fed at 0.08% of the diet DM, rumen protected choline (CHOL) fed at 60 g/d, and saline injections at d 0 and 7, 0.5X: TMR, MET, CHOL, 5 mg B12, and 80 mg folate at d 0 and 7, 1X: TMR, MET CHOL, 10 mg vitamin B12, and 160 mg folate at d 0 and 7, and 2X: TMR, MET, CHOL, 20 mg B12, and 320 mg folate at d 0 and 7. All heifers were estrus synchronized but not bred, and blood was collected on d 0, 2, 5, 7, 9, 12, and 14 of a synchronized estrous cycle. Heifers were slaughtered on d 14 of the estrous cycle for liver collection. Serum B12 concentrations were greater in the 0.5X, 1X, and 2X, compared with 0XNEG and 0XPOS on all days after treatment initiation (P < 0.0001). Serum folate concentrations were greater for the 2X treatment at d 5, 7, and 9 of the cycle compared with all other treatments (P ≤ 0.05). There were no differences (P ≥ 0.19) in hepatic methionine-cycle or choline analyte concentrations by treatment. Concentrations of hepatic folate cycle intermediates were always greater (P ≤ 0.04) in the 2X treatment compared with the 0XNEG and 0XPOS heifers. Serum methionine was greater (P = 0.04) in the 0.5X and 2X heifers compared with 0XNEG, and S-adenosylhomocysteine (SAH) tended (P = 0.06) to be greater in the 0.5X heifers and the S-adenosylmethionine (SAM):SAH ratio was decreased (P = 0.05) in the 0.5X treatment compared with the 0XNEG, 0XPOS, and 2X heifers. The hepatic transcript abundance of MAT2A and MAT2B were decreased (P ≤ 0.02) in the 0.5X heifers compared with the 0XNEG, 0XPOS, and 2X heifers. These data support that beef heifers fed rumen protected methionine and choline require 20 mg B12 and 320 mg folate once weekly to maintain increased concentrations of B12 and folate in serum. Furthermore, these data demonstrate that not all supplementation levels are equal in providing positive responses, and that some levels, such as the 0.5X, may result in a stoichiometric imbalance in the one-carbon metabolism pathway that results in a decreased SAM:SAH ratio.

The circadian rhythm of melatonin secretion in the pineal gland is highly conserved in vertebrates. Melatonin levels are always elevated at night. Acetylserotonin O-methyltransferase (ASMT) is the last enzyme in the regulation of melatonin biosynthesis (N-acetyl-5-hydroxytryptamine-melatonin). S-adenosylmethionine (SAM) is an important methyl donor in mammals and can be used as a substrate for the synthesis of melatonin. Methionine adenosyltransferase (MAT) catalyzes the synthesis of SAM from methionine and ATP and has a circadian rhythm. CircRNA is an emerging type of endogenous noncoding RNA with a closed loop. Whether circRNAs in the pineal gland can participate in the regulation of melatonin synthesis by binding miRNAs to target mat2a as part of the circadian rhythm is still unclear. In this study, we predicted the targeting relationship of differentially expressed circRNAs, miRNAs and mRNAs based on the results of rat pineal RNA sequencing. Mat2a siRNA transfection confirmed that mat2a is involved in the synthesis of melatonin. Circ-ERC2 and miR-125a-5p were screened out by software prediction, dual-luciferase reporter experiments, cell transfection, etc. Finally, we constructed a rat superior cervical ganglionectomy model (SCGx), and the results showed that circ-ERC2 could participate in the synthesis of melatonin through the miR-125a-5p/MAT2A axis. The results of the study revealed that circ-ERC2 can act as a molecular sponge of miR-125a-5p to regulate the synthesis of melatonin in the pineal gland by targeting mat2a. This experiment provides a basis for research on the circadian rhythm of noncoding RNA on pineal melatonin secretion.

The RNA methylation of adenosine at the N6-position (m6A) has attracted significant attention because of its abundance and dynamic nature. It accounts for more than 80% of all RNA modifications present in bacteria and eukaryotes and regulates crucial aspects of RNA biology and gene expression in numerous biological processes. The majority of m6A found in mammals is deposited by a multicomponent complex formed between methyltransferase-like (METTL) proteins METTL3 and METTL14. In the last few years, the list of m6A writers has grown, resulting in an expansion of our understanding of the importance of m6A and the methylation machinery. The characterization of the less familiar family member METTL16 has uncovered a new function of the m6A methylation apparatus, namely the fine-tuning of the cellular levels of the major methyl donor S-adenosylmethionine (SAM). METTL16 achieves this by adjusting the levels of the enzyme that synthesizes SAM in direct response to fluctuations in the SAM availability. This review summarizes recent progress made in understanding how METTL16 can sense and relay metabolic information and considers the wider implications. A brief survey highlights similarities and differences between METTL16 and the better-known METTL3/14 complex, followed by a discussion of the target specificity, modes of action and potential roles of METTL16.

Deoxyribonucleic acid-protein (DNAP) of the cell nucleus was purified from developing wheat (Triticum aestivum L.) embryo cells under drought stress, with two cultivars differing in drought tolerance as experimental materials - Longmai No. 079 (drought-tolerant) and Wanmai No. 52 (drought-sensitive). Levels of polyamines (PAs) non-covalently conjugated to the DNA and covalently conjugated to the proteins of DNAP were detected. After soil drought treatment for 10 days, in drought-tolerant Longmai No. 079, the increases in the levels of spermine and spermidine non-covalently conjugated to DNA of DNAP were more statistically significant (P<0.05) than in drought-sensitive Wanmai No. 52. Treatment of Wanmai No. 52 with exogenous Spm could not only enhance the tolerance of the cultivar to drought stress, as judged by flag leaf water content, plasma membrane permeability and grain growth, but also elevate the levels of spermine and spermidine noncovalently conjugated to the DNA of the cultivar. On the contrary, treatment of Longmai No. 079 with methylglyoxyl-bis guanylhydrazone, an inhibitor of S-adenosylmethionine decarboxylase, could significantly (P<0.05) aggravate the drought stress to this cultivar, accompanied by a marked decreases in the levels of spermine and spermidine non-covalently conjugated to the DNA of the cultivar. On the other hand, the content of putrescine covalently conjugated to the proteins of DNAP rose more markedly (P<0.05) in Longmai No. 079 than in Wanmai No. 52. The transglutaminase inhibitor, o-phenanthrolin, could markedly reduce the drought-induced increase in the level of putrescine covalently conjugated to the proteins of DNAP and aggravate drought stress to the two cultivars. Collectively, it could be inferred that spermine and spermidine non-covalently conjugated to the DNA and putrescine covalently conjugated to the proteins of DNAP in the developing grain embryo cell nucleus might enhance the tolerance of wheat plants to soil drought.

S-Adenosylmethionine (SAM) is a crucial small-molecule metabolite widely used in food and medicine. The development of high-throughput biosensors for SAM biosynthesis can significantly improve the titer of SAM. This paper constructed a synthetic transcription factor (TF)-based biosensor for SAM detecting in Saccharomyces cerevisiae. The synthetic TF, named MetJ-hER-VP16, consists of an Escherichia coli-derived DNA-binding domain MetJ, GS linker, the human estrogen receptor binding domain hER, and the viral activation domain VP16. The synthetic biosensor is capable of sensing SAM in a dose-dependent manner with fluorescence as the output. Additionally, it is tightly regulated by the inducer SAM and β-estradiol, which means that the fluorescence output is only available when both are present together. The synthetic SAM biosensor could potentially be applied for high-throughput metabolic engineering and is expected to improve SAM production.

Cupin proteins are involved in plant growth and development as well as in response to various stresses. Here, a total of 173 Cupin genes were identified in Brassica napus, and their molecular evolution and expression patterns were analyzed. These genes were classified into ten groups. Motif and exon-intron structure indicated a high degree of conservation within each group during evolution. BnaCupins were distributed on 19 chromosomes and their expansion is mainly contributed by whole-genome duplication (WGD) and segmental duplication events. BnaCupins have undergone severe purifying selection during a long evolutionary process. Meanwhile, some positive selection sites were identified. Expression patterns and cis-element analysis indicated that BnaCupins play significant roles in plant growth and stress responses. In addition, the expression levels of some BnCupins were significantly altered when treated with different conditions (cold, salt, drought, IAA, ABA, and 6-BA). Some BnaCupin interacting proteins, such as glycosyl hydrolase5 (GHs5), carbohydrate kinase (CHKs), ATP-dependent 6-phosphofructokinase (ATP-PFK), S-adenosylmethionine synthase (S-MAT), and aldolase class II (ALD II), were identified by the protein-protein interaction network. It will contribute to enriching our knowledge of the Cupin gene family in B. napus and provide a basis for further studies of their functions.