Enhanced levels of methionine and cysteine in transgenic alfalfa (Medicago sativa L.) plants over-expressing the Arabidopsis cystathionine gamma-synthase gene

With the aim of increasing the methionine level in alfalfa (Medicago sativa L.) and thus improving its nutritional quality, we produced transgenic alfalfa plants that expressed the Arabidopsis cystathionine gamma-synthase (AtCGS), the enzyme that controls the synthesis of the first intermediate metabolite in the methionine pathway. The AtCGS cDNA was driven by the Arabidopsis rubisco small subunit promoter to obtain expression in leaves. Thirty transgenic plants were examined for the transgene protein expression, and four lines with a high expression level were selected for further work. In these lines, the contents of methionine, S-methylmethionine (SMM), and methionine incorporated into the water-soluble protein fraction increased up to 32-fold, 19-fold, and 2.2-fold, respectively, compared with that in wild-type plants. Notably, in these four transgenic lines, the levels of free cysteine (the sulphur donor for methionine synthesis), glutathione (the cysteine storage and transport form), and protein-bound cysteine increased up to 2.6-fold, 5.5-fold, and 2.3-fold, respectively, relative to that in wild-type plants. As the transgenic alfalfa plants over-expressing AtCGS had significantly higher levels of both soluble and protein-bound methionine and cysteine, they may represent a model and target system for improving the nutritional quality of forage crops.     

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine

Aims:  To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma.
Methods and Results:  Probiotic strains ( Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H₂S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs ( P  < 0·05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses.
Conclusions:  Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species.
Significance and Importance of the Study:  This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.     

Lysine enhances methionine content by modulating the expression of S-adenosylmethionine synthase

Lysine and methionine are two essential amino acids whose levels affect the nutritional quality of cereals and legume plants. Both amino acids are synthesized through the aspartate family biosynthesis pathway. Within this family, lysine and methionine are produced by two different branches, the lysine branch and the threonine-methionine branch, which compete for the same carbon/amino substrate. To elucidate the relationship between these biosynthetic branches, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme dihydrodipicolinate synthase (DHPS) and contains a significantly higher level of lysine, and a second that overexpresses Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Significantly higher levels of methionine and its metabolite, S-methylmethionine (SMM), accumulated in the newly produced plants compared with plants overexpressing AtCGS alone, while the level of lysine remained the same as in those overexpressing DHPS alone. The increased levels of methionine and SMM were correlated with increases in the mRNA and protein levels of AtCGS and a reduced mRNA level for the genes encoding S-adnosylmethionine (SAM) synthase, which converts methionine to SAM. Reduction in SAMS expression level leads most probably to the reduction of SAM found in plants that feed with lysine. As SAM is a negative regulator of CGS, this reduction leads to higher expression of CGS and consequently to an increased level of methionine. Elucidating the relationship between lysine and methionine synthesis may lead to new ways of producing transgenic crop plants containing increased methionine and lysine levels, thus improving their nutritional quality.     

A sulfur amino acid deficiency changes the amino acid composition of body protein in piglets

Experiments carried out to determine the amino acid requirement in growing animals are often based on the premise that the amino acid composition of body protein is constant. However, there are indications that this assumption may not be correct. The objective of this study was to test the effect of feeding piglets a diet deficient or not in total sulfur amino acids (TSAA; Met + Cys) on nitrogen retention and amino acid composition of proteins in different body compartments. Six blocks of three pigs each were used in a combined comparative slaughter and nitrogen balance study. One piglet in each block was slaughtered at 42 days of age, whereas the other piglets received a diet deficient or not in TSAA for 19 days and were slaughtered thereafter. Two diets were formulated to provide either 0.20% Met and 0.45% TSAA (on a standardized ileal digestible basis) or 0.46% Met and 0.70% TSAA. Diets were offered approximately 25% below ad libitum intake. At slaughter, the whole animal was divided into carcass, blood, intestines, liver, and the combined head, tail, feet and other organs (HFTO), which were analyzed for nitrogen and amino acid contents. Samples of the longissimus muscle (LM) were analyzed for myosin heavy chain (MyHC) and actin contents. Nitrogen retention was 20% lower in piglets receiving the TSAA-deficient diet (P < 0.01). In these piglets, the nitrogen content in tissue gain was lower in the empty body, carcass, LM and blood (P < 0.05) or tended to be lower in HFTO (P < 0.10), but was not different in the intestines and liver. The Met content in retained protein was lower in the empty body, LM and blood (P < 0.05), and tended to be lower in the carcass (P < 0.10). The Cys content was lower in LM, but higher in blood of piglets receiving the TSAA-deficient diet (P < 0.05). Skeletal muscle appeared to be affected most by the TSAA deficiency. In LM, the Met content in retained protein was reduced by 12% and total Met retention by more than 60%. The MyHC and actin contents in LM were not affected by the TSAA content of the diet. These results show that a deficient TSAA supply affects the amino acid composition of different body proteins. This questions the use of a constant ideal amino acid profile to express dietary amino acid requirements, but also illustrates the plasticity of the animal to cope with nutritional challenges.     

Effect of dietary sulfur amino acids on the taurine content of rat tissues

Summary.  The effect of dietary sulfur amino acids on the taurine content of rat blood and tissues was investigated. Three types of diet were prepared for this study: a low-taurine diet (LTD), normal taurine diet (NTD; LTD + 0.5% Met), and high-taurine diet (HTD; LTD + 0.5% Met + 3% taurine). These diets had no differing effect on the growth of the rats. The concentration of taurine in the blood from the HTD- and NTD-fed rats was respectively 1,200% and 200% more than that from LTD-. In such rat tissues as the liver, the taurine content was significantly affected by dietary sulfur amino acids, resulting in a higher content with HTD and lower content with LTD. However, little or no effect on taurine content was apparent in the heart or eye. The activity for taurine uptake by the small intestine was not affected by dietary sulfur amino acids. The expression level of taurine transporter mRNA was altered only in the kidney under these dietary conditions: a higher expression level with LTD and lower expression level with HTD. Received January 8, 2002 Accepted January 18, 2002 Published online August 20, 2002 Authors' address: Dr. Hideo Satsu, Laboratory of Food Chemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, Fax: +81-3-5841-8026 E-mail: asatsu@mail.ecc.u-tokyo.ac.jp Abbreviations: HTD, high-taurine diet; NTD, normal taurine diet; LTD, low-taurine diet; TAUT, taurine transporter; CSA, cysteine sulfinate; CDO, cysteine dioxygenase; CSAD, cysteine sulfinate decarboxylase; PBS, phosphate-buffered saline; DIDS, 4,4′-diisothiocyanostilbene-2′,2′-disulfonic acid     

Follow the leader: preference for specific amino acids directly following the initial methionine in proteins of different organisms

It is well established that the vast majority of proteins of all taxonomical groups and species are initiated by an AUG codon, translated into the amino acid methionine (Met). Many attempts were made to evaluate the importance of the sequences surrounding the initiation codon, mostly focusing on the RNA sequence. However, the role and importance of the amino acids following the initiating Met residue were rarely investigated, mostly in bacteria and fungi. Herein, we computationally examined the protein sequences of all major taxonomical groups represented in the Swiss-Prot database, and evaluated the preference of each group to specific amino acids at the positions directly following the initial Met. The results indicate that there is a species-specific preference for the second amino acid of the majority of protein sequences. Interestingly, the preference for a certain amino acid at the second position changes throughout evolution from lysine in prokaryotes, through serine in lower eukaryotes, to alanine in higher plants and animals.     

Synthesis of novel acetylene-containing amino acids

Summary.  Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed. The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed ring-closing alkyne metathesis reactions. Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002 Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW). Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands, E-mail: rutjes@sci.kun.nl  2, selected data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.  Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH₂) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature, trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane (69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc. CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was extracted using heptane, washed with brine, dried (MgSO₄) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; ¹H NMR (300 MHz, CDCl₃) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C₁₇H₂₇NO₄ 309.1940, found 309.1937.  A solution of the tungsten catalyst (7 mg, 10 mol%) in C₆H₅Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C₆H₅Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]_D =–14.6 (c = 1, CH₂Cl₂); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for C₁₈H₂₈N₂O₅     

Cysteine restriction‐specific effects of sulfur amino acid restriction on lipid metabolism

Decreasing the dietary intake of methionine exerts robust anti‐adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti‐adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine‐ and cysteine‐titrated diets, we demonstrate that the anti‐adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non‐glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck‐M. In mice, the magnitude of SAAR‐induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age‐at‐onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross‐sectional epidemiological study. Controlled feeding of low‐SAA, high‐polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.     

Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants

Summary. Serine acetyltransferase (SATase) and cysteine synthase (O-acetylserine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by l-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by l-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for l-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by l-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F₁) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 μL L⁻¹ sulfur dioxide, both the cysteine and glutathione contents in leaves of F₁ plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants. Received November 27, 2001 Accepted December 21, 2001     

Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis

TheSalmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of theS. typhimurium andEscherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. Thein vitro andin vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. Thecys M gene ofS. typhimurium possessed a GUG initiation codon, like itsE. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteinein vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.     

Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Summary Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)-α-hydroxymethyltyrosine in position 1 and (R)- and (S)-α-hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that α-hydroxymethyl substitution of amino acid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

Radioactive methionine: determination, and distribution of radioactivity in the sulfur, methyl and 4-carbon moieties

A simple and inexpensive method is described for isolation and determination of [14C]methionine in the non-protein fraction of tissues extensively labeled with 14C. The effectiveness of the method was demonstrated by isolation of non-protein [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from the plant Lemna which had been grown for a number of generations on [U-14C]sucrose and contained a 2000-fold excess of 14C in undefined non-protein compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. This method also offers an attractive alternative to the use of more expensive and sophisticated equipment for assay of radioactive methionine under less demanding conditions. An advantage is that the isolated methioninecarboxymethylsulfonium salt is readily degraded to permit separate determination of radioactivity in the 4-carbon, methyl and sulfur moieties of methionine. During this work, a facile labilization of 3H attached to the (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt was observed. This labilization is ascribed to formation of a sulfur ylid.     

In vivo analysis of various substrates utilized by cystathionine gamma-synthase and O-acetylhomoserine sulfhydrylase in methionine biosynthesis

To gain insight into the evolution of the methionine biosynthesis pathway, in vivo complementation tests were performed. The substrate specificity of three enzymes that intrinsically use different homoserine-esterified substrates and have different sulfur assimilation pathways was examined: two cystathionine gamma-synthases (the Escherichia coli enzyme that naturally utilizes O-succinylhomoserine [OSH]) and the Arabidopsis thaliana enzyme that naturally exploits O-phosphohomoserine [OPH]. Both of these act through the transsulfuration pathway. The third enzyme investigated was O-acetylhomoserine (OAH) sulfhydrylase of Leptospira meyeri, representing the enzyme that utilizes OAH and operates through the direct sulfhydrylation pathway. All the three enzymes were able to utilize OSH and OAH as substrates, with different degrees of efficiency, but only the plant enzyme was able to utilize OPH as a substrate. In addition to their inherent activity in the transsulfuration pathway, the two cystathionine gamma-synthases were also capable of acting in the direct sulfhydrylation pathway. Based on the phylogenic tree and the results of the complementation tests, we suggest that the ancestral gene was able to act as OAH or OSH sulfhydrylase. In some bacteria and plants, this ancient enzyme most probably evolved into a cystathionine gamma-synthase, thereby maintaining the ability to utilize various homoserine-esterified substrates, as well as various sulfur sources, and thus keeping the multisubstrate specificity of its ancestor. In some organisms, this ancestral gene probably underwent a duplication event, which resulted in a cystathionine gamma-synthase and a separate OAH or OSH sulfhydrylase. This led to the development of two parallel pathways of methionine biosynthesis, transsulfuration and direct sulfhydrylation, in these organisms. Although both pathways exist in several organisms, most seem to favor a single specific pathway for methionine biosynthesis in vivo.