S-adenosyl methionine requiring mutants in Saccharomyces cerevisiae: evidences for the existence of two methionine adenosyl transferases.

Summary Mutants requiring S-adenosyl methionine (SAM) for growth have been selected in Saccharomyces cerevisiae. Two classes of mutants have been found. One class corresponds to the simultaneous occurrence of mutations at two unlinked loci SAM1 and SAM2 and presents a strict SAM requirement for growth on any medium. The second class corresponds to special single mutations in the gene SAM2 which lead to a residual growth on minimal medium but to normal growth on SAM supplemented medium or on a complex medium like YPGA not containing any SAM. These genetic data can be taken as an indication that Saccharomyces cerevisiae possesses two isoenzymatic methionine adenosyl transferases (MAT). In addition, SAM1 and SAM2 loci have been identified respectively with the ETH-10 and ETH2 loci previously described.Biochemical evidences corroborate the genetic results. Two MAT activities can be dissociated in a wild type extract (MATI and MATII) by DEAE cellulose chromatography. Mutations at the SAM1 locus lead to the absence or to the modification of MATII whereas mutations at the SAM2 locus lead to the absence or to the modification of MATI. Moreover, some of our results seem to show that MATI and MATII are associated in vivo.     

The synthesis of the two S-adenosyl-methionine synthetases is differently regulated in Saccharomyces cerevisiae

Summary S-adenosyl-l-methionine (AdoMet) is synthesized by transfer of the adenosyl moiety of ATP to the sulfur atom of methionine. This reaction is catalysed by AdoMet synthetase. In all eukaryotic organisms studied so far, multiple forms of AdoMet synthetases have been reported and from their recent study, it appears that AdoMet synthetase is an exceptionally well conserved enzyme through evolution. In Saccharomyces cerevisiae, we have demonstrated the existence of two AdoMet synthetases encoded by genes SAM1 and SAM2. Yeast, which is able to concentrate exogenously added AdoMet, is thus a particularly useful biological system to understand the role and the physiological significance of the preservation of two almost identical AdoMet synthetases. The analysis of the expression of the two SAM genes in different genetic backgrounds during growth under different conditions shows that the expression of SAM1 and SAM2 is regulated differently. The regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, where as SAM2 shows a specific pattern of regulation. A careful analysis of the expression of the two genes and of the variations in the methionine and AdoMet intracellular pools during the growth of different strains lead us to postulate the existence of two different AdoMet pools, each one suppplied by a different AdoMet synthetase but in equilibrium with each other. This could be a means of storing AdoMet whenever this metabolite is overproduced, thus avoiding the degradation of a metabolite the synthesis of which is energetically expensive.     

Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation

Aims: S‐adenosyl‐l ‐methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Methods and Results: Through spaceflight culture, a SAM‐accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one‐factor‐at‐a‐time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76 g l^?1 of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64 g l^?1 of SAM was produced in fermenter cultivation. Conclusions: A new SAM‐accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Significance and Impact of the Study: Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76 g l^?1 of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84 h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications.     

S-Adenosyl Methionine-Mediated Repression of Methionine Biosynthetic Enzymes in Saccharomyces cerevisiae

S-adenosylmethionine (SAM) has been shown to provoke repression of some methionine-specific enzymes in wild-type cells, namely, adenosine triphosphate sulfurylase, sulfite reductase, and homocysteine synthetase. Repressive effects observed in SAM-supplemented cultures should be due to SAM per se, since the intracellular pool of SAM increases while the intracellular pool of methionine remains low and constant. Derepression brought about by methionine limitation is accompanied by a severe decrease in SAM as well as methionine pool sizes, although methionine adenosyl transferase is slightly derepressed. Different hypotheses have been considered to account for the previously reported implication of methionyl transfer ribonucleic acid and the presently reported SAM effects in this regulatory process.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Deficiency in methionine adenosyltransferase resulting in limited repressibility of methionine biosynthetic enzymes in Aspergillus nidulans

Summary In Aspergillus nidulans methionine can be metabolized to cysteine. Mutants blocked in this pathway were selected and divided into three groups representing three separate loci: mecA, mecB and mecC. mecC13 mutant possesses a low level of methionine adenosyltransferase and shows a limited extent of methionine-caused repression of three enzymes of the methionine biosynthetic pathway: sulfate permease, sulfite reductase and 0-acetylhomoserine sulfhydrylase. Intracellular pools of methionine do not differ markedly in the mutant and in wild type, while the S-adenosylmethionine (SAM) pool is decreased in the mutant. Methionine adenosyltransferase was found to be inducible by methionine, SAM is postulated to be involved in regulation of methionine biosynthetic enzymes in A. nidulans. Differences in regulation of methionine biosynthesis in A. nidulans, Escherichia coli and Saccharomyces cerevisiae are discussed.     

Refolding of a fully functional flavivirus methyltransferase revealed that S‐adenosyl methionine but not S‐adenosyl homocysteine is copurified with flavivirus methyltransferase

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2′‐O positions of the viral RNA cap by using S‐adenosyl‐l ‐methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by‐product S‐adenosyl‐l ‐homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM‐analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.     

Effects of Regulatory Mutations upon Methionine Biosynthesis in Saccharomyces cerevisiae: Loci eth2-eth3-eth10

The effects of mutations occurring at three independent loci, eth2, eth3, and eth10, were studied on the basis of several criteria: level of resistance towards two methionine analogues (ethionine and selenomethionine), pool sizes of free methionine and S-adenosyl methionine (SAM) under different growth conditions, and susceptibility towards methionine-mediated repression and SAM-mediated repression of some enzymes involved in methionine biosynthesis (met group I enzymes). It was shown that: (i) the level of resistance towards both methionine analogues roughly correlates with the amount of methionine accumulated in the pool; (ii) the repressibility of met group I enzymes by exogenous methionine is either abolished or greatly lowered, depending upon the mutation studied; (iii) the repressibility of the same enzymes by exogenous SAM remains, in at least three mutants studied, close to that observed in a wild-type strain; (iv) the accumulation of SAM does not occur in the most extreme mutants either from endogenously overproduced or from exogenously supplied methionine: (v) the two methionine-activating enzymes, methionyl-transfer ribonucleic acid (tRNA) synthetase and methionine adenosyl transferase, do not seem modified in any of the mutants presented here; and (vi) the amount of tRNA^met and its level of charging are alike in all strains. Thus, the three recessive mutations presented here affect methionine-mediated repression, both at the level of overall methionine biosynthesis which results in its accumulation in the pool, and at the level of the synthesis of met group I enzymes. The implications of these findings are discussed.     

Methionine is required for cAMP‐PKA‐mediated morphogenesis and virulence of Candida albicans

Candida albicans is a major human fungal pathogen, causing superficial, as well as life‐threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast‐to‐hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine‐induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S‐adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino‐propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine‐induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP‐PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.     

The effects of 5‐azacytidine and cadmium on global 5‐methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S‐adenosyl‐L‐methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5‐azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S‐adenosylhomocysteine, 5′‐methylthioadenosine and global 5‐methylcytosine (5‐mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5‐mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5‐azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5‐mC content of C. reinhardtii was 1%–1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S‐adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.     

The effects of L-dopa on the activity of methionine adenosyltransferase: Relevance to L-dopa therapy and tolerance

L-dopa, the major treatment for Parkinson's disease (PD), depletes S-adenosyl-L-methionine (SAM). Since SAM causes PD-like symptoms in rodents, the decreased efficacy of chronic L-dopa administered to PD patients may result from a rebound increase in SAM via methionine adenosyl transferase (MAT), which produces SAM from methionine and ATP. This was tested by administering intraperitoneally saline, or L-dopa to mice and assaying for brain MAT activity. As compared to controls, L-dopa (100 mg/kg) treatments of 1 and 2 times per day for 4 days did not significantly increase MAT activity. However, treatments of 1 and 2 times per day for 4 and 8 days did significantly increase the activity of MAT by 21.38% and 28.37%, respectively. These results show that short interval, chronic L-dopa treatments significantly increases MAT activity, which increases the production of SAM. SAM may physiologically antagonize the effects of L-dopa and biochemically decrease the concentrations of L-dopa and dopamine. Thus, an increase in MAT may be related to the decreased efficacy of chronic L-dopa therapy in PD.     

TheSAM2 gene product catalyzes the formation of S-adenosyl-ethionine from ethionine inSaccharomyces cerevisiae

Ethionine is the toxic S-ethyl analog of the essential amino acid methionine. Whereas in prokaryotes the ethionine just competes with the methionine, in eukaryotes it can also be transformed into S-adenosyl-ethionine (Ado-Eth), competing with the S-adenosyl-methionine (Ado-Met). When the Ado-Met synthetase activity was studied in strains defective in either of the two isoenzymes, the one coded by theSAM1 gene was totally unable to convert ethionine into Ado-Eth and was inhibited by the analog, whereas the enzyme coded by theSAM2 gene was able to bind ethionine and was not inhibited by it. This has allowed the development of a procedure to measure Ado-Met synthetase and differentiate between the two isoenzymes present inSaccharomyces cerevisiae.     

Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae

Summary We have investigated the physiological conditions under which meiosis and the ensuing sporulation of Saccharomyces cerevisiae are initiated. Initiation of sporulation occurs in response to carbon, nitrogen, phosphorus, or sulfur deprivation, and also, when met auxotrophs are partially starved for methionine, but not after starvation of other amino acid auxotrophs. It also occurs after partial starvation of pur or gua auxotrophs for guanine but not after starvation of ura auxotrophs for uracil. Under all these sporulation conditions the concentrations of both guanine nucleotides (GTP) and S-adenosylmethionine (SAM) decrease whereas those of other nucleotides show no trend. We show that the decrease of guanine nucleotides is essential for the initiation of meiosis and sporulation: when a gua auxotroph, also lacking one of the two SAM synthetases, is starved for guanine but supplemented with 0.1 mM methionine, GTP decreases while SAM slightly increases and yet the cells sporulate.     

The synthesis of S-adenosylmethionine by mutants with defects in S-adenosylmethionine synthetase

Summary Some metK mutants of Salmonella typhimurium with constitutive methionine biosynthesis have no detectable S-adenosylmethionine (SAM) synthetase, the enzyme which converts methionine to SAM, the postulated corepressor of the methionine pathway. However these mutants are not auxotrophic for SAM, an essential compound for many reactions. Here it is shown that these mutants have normal functioning of pathways involving SAM and do in fact produce SAM at as high levels as wild-type. Also, SAM synthetase is shown to be dispensible for growth but not for methionine regulation. These results indicate that there is another route of SAM synthesis independent of SAM synthetase. This route probably also uses methionine as substrate as metK mutants are shown to convert methionine to SAM as efficiently as analogous non-metK strains. The existence of a second route of SAM synthesis makes it necessary to postulate a compartmentalization of SAM made via the SAM synthetase reaction from SAM made in any other way to explain the reduced ability of metK mutants to repress methionine biosynthesis.     

S-adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium

Summary In wild-type bacteria, S-adenosylmethionine (SAM) synthetase activity was repressed by growth in methionine. MetJ regulatory mutants had elevated activities which did not show this repression. Two metK mutants with normal regulation of the methionine biosynthetic enzymes had elevated Km's (methionine) for SAM synthetase while five metK mutants with constitutive methionine enzymes showed no measurable SAM synthetase activity. One mutant, metK ^X 721, similar in phenotype to these five had a wild-type level of SAM synthetase in conditions where SAM decarboxylase activity was blocked. By using an F-factor carrying the metK region of the genome, this mutant was shown to complement six other metK mutants.These results indicate that SAM or a derivative of it, rather than methionine itself, is the co-repressor of the methionine system, regulatory abnormalities resulting from the absence or reduction of the amount of SAM formed by the SAM synthetase reaction. As SAM is essential for bacteria it is likely that there is some alternative biosynthetic route for SAM.     

S-Adenosylmethionine (SAM)-Accumulating Sake Yeast Suppresses Acute Alcohol-Induced Liver Injury in Mice

The suppressive effects on acute alcoholic liver injury of S-adenosylmethionine (SAM) and the sake yeast, Saccharomyces cerevisiae Kyokai No. 9, have been shown previously. To enhance the suppression of acute alcoholic liver injury by sake yeast, we prepared SAM-accumulating sake yeast (SAM yeast). Male C57BL/6 mice that had been fed on a diet containing 0.25% SAM yeast or sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed on the SAM yeast, the ethanol-induced increases in both triglyceride (TG) and alanine aminotransferase (ALT) were significantly repressed. In addition, the SAM yeast-fed mice did not show an ethanol-induced decrease in hepatic SAM level, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by the SAM yeast. These results suggest that the SAM yeast had a stronger effect suppressing acute alcoholic liver injury in mice than the sake yeast.     

Overexpression of S‐adenosyl‐l‐methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism

S‐adenosyl‐l ‐methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1‐overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na⁺ absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.     

Viperin is an iron‐sulfur protein that inhibits genome synthesis of tick‐borne encephalitis virus via radical SAM domain activity

Viperin is an interferon‐induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S‐adenosyl‐l ‐methionine (SAM) domain which has been shown in vitro to hold a [4Fe‐4S] cluster. We identified tick‐borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of ⁵⁵Fe, indicative for the presence of an Fe‐S cluster. Mutation of the cysteine residues ligating the Fe‐S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of ⁵⁵Fe. Mutants lacking the extreme C‐terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe‐4S] cluster assembly, and acts through an enzymatic, Fe‐S cluster‐ and SAM‐dependent mechanism to inhibit viral RNA synthesis.