Isolation and Characterization of S-Adenosylmethionine-requiring Mutants and Role of S-Adenosylmethionine in the Regulation of Methionine Biosynthesis in Corynebacterium glutamicum

Using a minimal medium containing a methionine analog together with a small amount of S-adenosylmethionine (SAM), many SAM requiring mutants which responded only to SAM and not to methionine, S-adenosylhomocysteine, or homocysteine were efficiently isolated from Corynebacterium glutamicum TLD-140 after mutagenesis. Among them, SAM-14 and SAM-19 selected from selenomethionine resistant mutants were subjected to further investigation. Both mutants were unable to grow in a minimal medium and had no detectable activity of SAM synthetase. Both mutants acquired higher resistance to methionine hydroxamate and ethionine as well as to selenomethionine than TLD-140 and produced l-methionine in a medium.

Homoserine-O-transacetylase in SAM-19 was subject to full repression by the addition of excess SAM to the growth medium and was not repressed under SAM limitation, whereas addition of excess l-methionine under SAM limitation caused a partial repression of the enzyme. SAM synthetase as well as l-methionine biosynthetic enzymes in a methionine auxotroph of C. glutamicum was repressed by the addition of l-methionine to the growth medium.

These results suggest that SAM is implicated in the repression of l-methionine synthesizing enzymes in C. glutamicum.     

Selection of Methionine-Resistant Variant in Onobrychis viciaefolia Scop.

The variant cell line of Onobrychis viciaefolia Scop. (ONmetr) resistant to 80 mmol/L methionine was isolated from calli which was treated with NAN3. This ONmet cell line was induced to regenerate plantlets. After growing for 6 months on a medium without selection pressure, the ONmetr cell line was still highly resistant to methionine being 5.6-fold higher than that of the wild type. The variant cell line also expressed high level of cross-resistance to ethionine which was 6. b-fold higher than that of the wild type. The contents of total methionine (Met) ,lysine (Lys) ,threonine (Thr) in ONmetr calli were 4.00,1.09,1.50-fold respectively higher than those of the wild type. The contents of total Met、 Lys、 Thr、 Ile (isoleucine) in ONmetr regenerants were-2.0, 3.5,3. 5,2. 5-fold respectively higher than those of the wild type. Two new bands appeared in SDS-PAGE profile as well as in the superoxidase isoenzymes electrophoresis pattern of the soluble proteins of ONmetr calli, thus indicated that the variant had carried the products of the changed genes.     

Metabolism of Ethionine in Ethionine-Sensitive and Ethionine-Resistant Cells of the Enteric Yeast Candida slooffii

In a defined medium with added ethionine plus low methionine, phenylalanine, tryptophan, tyrosine, adenine, and additional methionine reversed inhibition of the enteric yeast Candida slooffii by ethionine. Isoleucine and 7-methylguanine restored half-maximal growth. Choline but not triethylcholine inhibited C. slooffii. 6-Mercaptopurine reversed ethionine inhibition and also synergistic inhibition by ethionine plus choline. Protection against ethionine by adenine plus aromatics was also evident with log-phase cells in the absence of methionine. Incorporation of ethionine-ethyl-1-(14)C by resting cells was partially inhibited by aromatic amino acids and methionine. Ethionine depressed incorporation of (3)H-phenylalanine but not of (3)H-adenine. Ethionine-resistant mutants were isolated which incorporated ethionine efficiently and degraded it to yet unidentified substances not including 5'-ethylthioadenosine. Ethionine-sensitive cells accumulated more S-adenosylethionine (SAE) than resistant mutants. Adenine was a good precursor of SAE. Radioactivity from ethionine-ethyl-1-(14)C was recovered from cell fractions of ethionine-sensitive cells with the following distribution: cold trichloroacetic acid-soluble > hot trichloroacetic acid-insoluble > lipids > deoxyribonucleic acid > ribonucleic acid. Total radioactivity recovered from ethionine-sensitive cells was twice as much as that from ethionine-resistant mutants.     

Control of Free Methionine Production in Wild Type and Ethionine-resistant Mutants of Chlorella sorokiniana

Mutants of Chlorella sorokiniana selected for resistance to the methionine analogue ethionine took up ethionine at the same rate as did the wild type strain. Cells of two ethionine-resistant mutants produced severalfold higher levels of free methionine and cysteine than did wild type cells.     

Methionine biosynthesis in Saccharomyces cerevisiae: Mutations at the regulatory locus ETH2

Summary Homoallelic and heteroallelic diploids involving the eth2-1, eth2-2 and eth2-7 alleles have been studied on the basis of several criteria used for the study of haploid strains: resistance towards ethionine, overproduction of either methionine or/and S-adenosylmethionine, repressibility of methionine biosynthetic enzymes. Complete recessivity of the three alleles over the wild type allele has been observed, when resistance and methionine synthesis are considered. However, with the eth2-2 allele, repressibility corresponds more to a dose effect of the ETH2 allele than to recessivity. The implications of these findings have been discussed. Results obtained for heteroallelic combinations show significant deviations from the expected values. These results have been interpreted as indicating possible interactions between two differently impaired products of gene ETH2. They render likely that the product of this gene is at least an homopolymer.     

The effect of amino acids in the asparagine family on the aspartate kinase and homoserine dehydrogenase of ethionine-resistant mutants of Pseudomonas putida

The activity of aspartate kinase and homoserin dehydrogenase from ethionine resistant mutants Pseudomonas putida 25 and 6 have been studied as affected by amino acids from the family of asparagine. They are characterized by a capacity to the surplus synthesis of methionine. It is shown that mutants have negative regulation of the level of activity of the studied enzymes. It is supposed that the mutations (or mutation) could take place which affected properties of enzymes, which participated directly in the biosynthesis of methionine, in the analogue resistant clones 25 and 6.     

Isolation and characterisation ofCandida sp. mutants enriched in S-adenosylmethionine (SAM)

The ability to accumulate S-adenosylmethionine (SAM) of 572 yeast strains isolated from environmental sources were surveyed. An S-adenosylmethionine enriching strain S42-12, identified asCandida sp., was chose to develop a SAM-accumulating mutant successfully. The final SAM-accumulating mutant strain YQ-5 was isolated by UV radiation or by NTG treatment using ethionine selection and nystatin selection method. The mutant strain YQ-5 accumulated 112.1 mg per gram biomass, was 3.14-fold higher than the original strain S42-12. When cultivated in the optimal medium with a favourable fermentation conditions, SAM content of the mutant strain reached at 1740 mg L^?1. Trend of SAM and ergosterol contents and methionine adenosyltransferase activity of SAM-accumulating mutants during fermentation were analysed. The results suggested that one of the reasons why the mutants accumulated SAM in significantly high amounts may be the lower consumption of SAM for ergosterol biosynthesis, other than improvement of methionine adenosyltransferase activity.     

苜蓿抗甲硫氨酸变异体的筛选

紫花苜蓿(Medicago sativa L.)下胚轴愈伤组织用NaN_3溶液诱变处理后,在含有全致死浓度甲硫氨酸的MS培养基上进行了6个月的连续筛选培养,获得了能抗100mmol/L甲硫氨酸的变异细胞系,并分化成再生植株。所获变异细胞系在脱离选择压力6个月后,对甲硫氨酸的抗性仍比对照高7.2倍,并表现出对乙硫氨酸的交叉抗性(为对照抗性的3.3倍)。抗性细胞系及其再生植株的甲硫氨酸、赖氨酸、苏氨酸和异亮氨酸含量均比对照有大幅度增加。抗性系的SDS-PAGE电泳图谱及过氧化物酶同工酶谱带均与对照有显著不同,并出现了新带,表明变异系已经产生变化了的基因产物。     

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.     

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.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Regulation of S-Adenosylmethionine Synthetase in Escherichia coli

Addition of methionine to the growth medium of Escherichia coli K-12 leads to a reduction in the specific activity of S-adenosylmethionine (SAM) synthetase. Thus the enzyme appears to be repressible rather than inducible. Mutant strains (probably metJ(-)) are constitutive for SAM synthetase as well as for the methionine biosynthetic enzymes, suggesting that the regulatory systems for these enzymes have at least some elements in common. Cells grown to stationary phase in complete medium, which have low specific activities of the enzymes, were routinely used for derepression experiments. The lag in growth and derepression when these cells are incubated in minimal medium is shortened by threonine. Ethionine, norleucine, and alpha-methylmethionine are poor substrates or nonsubstrates for SAM synthetase and are ineffective repressors. Selenomethionine, a better substrate for SAM synthetase than methionine, is also slightly more effective at repression than methionine. Although SAM is considered to be a likely candidate for the corepressor in the control of the methionine biosynthetic enzymes, addition of SAM to the growth medium does not cause repression. Measurement of SAM uptake shows that too little is taken into the cells to have a significant effect, even if it were active in the control system.     

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.     

Improving methionine and ATP availability by <Emphasis Type="Italic">MET6</Emphasis> and <Emphasis Type="Italic">SAM2</Emphasis> co-expression combined with sodium citrate feeding enhanced SAM accumulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

S-adenosyl-l-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.     

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.     

Reversion in expression of hypoxanthine-guanine phosphoribosyltransferase in 6-thioguanine resistant neuroblastoma: evidence for reduced enzyme levels associated with unaltered catalytic activity.

Five clones of mouse neuroblastoma cells able to grow in hypoxanthine-aminopterin-thymidine containing medium were isolated from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine-aminopterin-thymidine resistant revertant clone had 45-55% of wild-type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5-phosphoribosyl-1-pyrophosphate were similar in wild-type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild-type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross-reacting material in normal and revertant clones. The revertant clones had one-half the amounth of cross-reacting material present in wild-type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.     

High free-methionine and decreased lignin content result from a mutation in the Arabidopsis S-adenosyl-L-methionine synthetase 3 gene

As an approach to understand the regulation of methionine (Met) metabolism, Arabidopsis Met over-accumulating mutants were isolated based on their resistance to selection by ethionine. One mutant, mto3, accumulated remarkably high levels of free Met - more than 200-fold that observed for wild type - yet showed little or no difference in the concentrations of other protein amino-acids, such as aspartate, threonine and lysine. Mutant plants did not show any visible growth differences compared with wild type, except a slight delay in germination. Genetic analysis indicated that the mto3 phenotype was caused by a single, recessive mutation. Positional cloning of this gene revealed that it was a novel S-adenosylmethionine synthetase, SAMS3. A point mutation resulting in a single amino-acid change in the ATP binding domain of SAMS3 was determined to be responsible for the mto3 phenotype. SAMS3 gene expression and total SAMS protein were not changed in mto3; however, both total SAMS activity and S-adenosylmethionine (SAM) concentration were decreased in mto3 compared with wild type. Lignin, a major metabolic sink for SAM, was decreased by 22% in mto3 compared with wild type, presumably due to the reduced supply of SAM. These results suggest that SAMS3 has a different function(s) in one carbon metabolism relative to the other members of the SAMS gene family.     

Monoclonal antibodies to leucine enkephalin

Monoclonal antibodies to leucine enkephalin have been produced after fusion of mouse myeloma cells with spleen cells from hyper-immune mice. Hybrid clones 2D1 and SL1 were characterised using radioimmunoassay and an enzyme-linked immunosorbent assay. The antibody 2D1 was of low affinity and showed a maximum sensitivity of 0.1ng. The antibody binds equally well to the sulphated leucine enkephalin and to methionine enkephalin. It does not cross-react with dynorphin, methionine enkephalin-arg-phe or oxidised methionine enkephalin. The hybrid clone SL1 appears to be specific for leucine enkephalin. Preliminary immunocytochemical studies have shown that both antibodies bind specifically to leucine enkephalin in defined areas of the central nervous system.     

Isolation and characterization of cultured mouse T-lymphoma cells deficient in uridine-cytidine kinase.

Two clones were isolated from mutagenized mouse T-lymphoma cells (S49) which are over 90% deficient in uridine-cytidine kinase. The first clone, AU-200-1, was isolated in two steps by virtue of its resistance to 6-azauridine; whereas the second clone, FU3-70G, was isolated in three steps after exposure to three increasing concentrations of 5-fluorouracil. Extracts of both the AU-200-1 and the FU3-70G cell lines lacked over 90% of the capacity of those from wild type cells to phosphorylate either uridine or cytidine. Furthermore, the uptake of radioactive uridine and cytidine from the medium by intact AU-200-1 and FU3-70G cells was less than 5% of that found for intact wild type cells. By growth rate experiments, these uridine-cytidine kinase-deficient cell lines have altered sensitivities to the toxic pyrimidine analogs, 6-azauridine, 5-fluorouracil, and 5-fluorouridine and thus have been useful in elucidating the biochemical determinants involved in the metabolism of these compounds.     

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.