Metabolic Pathway of O-alkylhomoserine in Corynebacterium acetophilum

The metabolic pathway of O-alkylhomoserine in Corynebacterium acetophilum was determined using mutants with defects in methionine biosynthesis and purified O-acetylhomoserine sulfhydrylase. The mutant strain M–74, defective in homoserine transacetylase, utilized O-alkylhomoserines and O-acetylhomoserine instead of methionine, but strain M–933, lacking cystathionine γ-synthase, did not. The incorporation of radioactive O-etbylhomoserine into cells was inhibited competitively by O-acetylhomoserine. Analysis of autoradiograms of two-dimensional thin-layer chromatograms showed that labeled O-ethylhomoserine was converted to O-acetylhomoserine by strain M–933 and finally metabolized to methionine by strain M–74. Furthermore, results showed that O-acetylhomoserine was synthesized from O-ethylhomoserine and acetic acid by a reversible side reaction of purified O-acetylhomoserine sulfhydrylase of C. acetophilum. These findings show that O-alkylhomoserine is converted to O-acetylhomoserine and then metabolized to methionine via cystathionine and homocysteine in cells of C. acetophilum starved of methionine.     

Methionine Synthesis in Lemna: Inhibition of Cystathionine gamma-Synthase by Propargylglycine

Propargylglycine, vinylglycine, and cysteine each cause irreversible inactivations of cystathionine γ-synthase (and, in parallel, of O-phosphohomoserine sulfhydrylase) activities in crude extracts of Lemna paucicostata. Inactivation by propargylglycine or vinylglycine is completely prevented by 40 millimolar O-phospho- or O-succinyl-l-homoserine; that by cysteine is only partially prevented. Propargylglycine (PAG), the most potent of these inhibitors, causes rapid and drastic inactivation of both activities in intact Lemna. Studies of plants growing in steady states in the presence of various concentrations (0-150 nanomolar) of PAG showed that 16% of control activity is necessary and sufficient to maintain normal rates of growth and methionine biosynthesis, and that 10% of control activity is essential for viability. Addition of either 2 micromolar methionine or 31 micromolar cystine to growth medium containing 150 nanomolar PAG permits growth at 75 to 100% of control rates when enzyme activity is less than 10% of control. Whereas methionine presumably rescues by directly providing the missing metabolite, cystine may rescue by enhancing substrate accumulation and thereby promoting flux through residual cystathionine γ-synthase. The results indicate that the down-regulation of cystathionine γ-synthase to 15% of control which occurs when plants are grown in 2 micromolar methionine (Thompson, Datko, Mudd, Giovanelli Plant Physiol 69: 1077-1083), by itself, is not sufficient to reduce the rate of methionine biosynthesis.     

Sulfur amino acid metabolism in Schizosaccharomyces pombe: occurrence of two O-acetylhomoserine sulfhydrylases and the lack of the reverse transfulfuration pathway

Abstract The fission yeast Schizosaccharomyces pombe has a unique organization of sulfur amino acid metabolism: it has two distinct O -acetylhomoserine sulfhydrylases (homocysteine synthases). Similar to Enterobacteriaceae, S. pombe lacks cystathionine β-synthase and cystathionine γ-lyase - the enzymes of the reverse transsulfuration pathway, by which methionine is readily metabolized to cysteine - a likely effector in the sulfur metabolite repression system. Consequently no repression of sulfate assimilation is observed when methionine is added to the growth medium.     

Molecular characterization of a cystathionine beta-synthase gene,CBS1, in Magnaporthe grisea

CBS1 from Magnaporthe grisea is a structural and functional homolog of the cystathionine β-synthase (CBS) gene from Saccharomyces cerevisiae. Our studies indicated that M. grisea can utilize homocysteine and methionine through a CBS-independent pathway. The results also revealed responses of M. grisea to homocysteine that are reminiscent of human homocystinuria.     

Threonine Synthase of Lemna paucicostata Hegelm. 6746

Threonine synthase (TS) was purified approximately 40-fold from Lemna paucicostata, and some of its properties determined by use of a sensitive and specific assay. During the course of its purification, TS was separated from cystathionine γ-synthase, establishing the separate identity of these enzymes. Compared to cystathionine γ-synthase, TS is relatively insensitive to irreversible inhibition by propargylglycine (both in vitro and in vivo) and to gabaculine, vinylglycine, or cysteine in vitro. TS is highly specific for O-phospho-l-homoserine (OPH) and water (hydroxyl ion). Nucleophilic attack by hydroxyl ion is restricted to carbon-3 of OPH and proceeds sterospecifically to form threonine rather than allo-threonine. The K_m for OPH, determined at saturating S-adenosylmethionine (AdoMet), is 2.2 to 6.9 micromolar, two orders of magnitude less than values reported for TS from other plant tissues. AdoMet markedly stimulates the enzyme in a reversible and cooperative manner, consistent with its proposed role in regulation of methionine biosynthesis. Cysteine (1 millimolar) caused a slight (26%) reversible inhibition of the enzyme. Activities of TS isolated from Lemna were inversely related to the methionine nutrition of the plants. Down-regulation of TS by methionine may help to limit the overproduction of threonine that could result from allosteric stimulation of the enzyme by AdoMet.     

Intracellular localization of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves

The intracellular localization of several aspartate pathway enzymes has been studied in pea (Pisum sativum cv Feltham First) and barley (Hordeum vulgare cv Julia) leaves. Protoplast lysates were fractionated by differential or sucrose density gradient centrifugation, in media optimized for each enzyme. The results show that aspartate kinase, homoserine kinase, threonine synthase, and cystathionine γ-synthase are confined to the chloroplast. Cystathionine β-lyase appears to be present in several fractions, though more than 50% of the total activity is associated with the chloroplasts. In contrast, neither methionine synthase nor methionine adenosyl-transferase were significantly associated with chloroplasts, and only a small proportion of the methionine synthase was associated with the mitochondrial fraction. Methionine adenosyltransferase, and hence S-adenosylmethionine synthesis, is not found in any organelle fraction. The conclusion is that whereas threonine, like lysine, is synthesized only in the chloroplast, the last step in methionine biosynthesis occurs largely in the cytoplasm.     

Methionine biosynthesis in higher plants: biochemical and molecular characterization of the transsulfuration pathway enzymes

In plants, the transfer of the sulfur atom between cysteine and homocysteine, the direct precursor of methionine, is ensured by two chloroplastic enzymes, cystathionine γ-synthase and cystathionine β-lyase. These proteins have been purified to homogeneity from spinach chloroplasts and their biochemical properties determined. Cystathionine γ-synthase and cystathionine β-lyase are tetramers and are typical pyridoxal 5′-phosphate-dependent proteins. These enzymes are targets for the potent inhibitors of methionine synthesis that are lethal for plants. An Arabidopsis thaliana cDNA encoding chloroplastic cystathionine β-lyase was isolated by functional complementation of a bacterial mutant and cloned in a pET expression vector in order to transform Escherichia coli cells. Preliminary observations of the active site of the purified recombinant enzyme have been performed by characterization of the interaction between i) pyridoxal 5′-phosphate and the polypeptide chain, and ii) the active site-directed inhibitor aminoethoxyvinylglycine and the bound cofactor. This study will be developed further by crystallographic analyses.     

Molecular and Functional Analyses of the metC Gene of Lactococcus lactis, Encoding Cystathionine β-Lyase

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the metC gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via α,γ elimination to form volatile aroma compounds.     

Effects of Orthophosphate and Adenosine 5'-Phosphate on Threonine Synthase and Cystathionine gamma-Synthate of Lemna paucicostata Hegelm. 6746

Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine γ-synthase less strongly. AMP is an extremely potent and structurally specific inhibitor of threonine synthase. Each inhibition progressively decreases with increasing concentrations of O-phosphohomoserine (OPH). To study the in vivo effects of these inhibitions, Lemna was grown with a range of Pi concentrations. A 25,000-fold increase in Pi concentration in the culture medium caused an increase of only 6-fold in total phosphorus of the plants. This is explained by the fact that a high affinity Pi uptake system is selectively down-regulated during growth with high concentrations of Pi. Pi and AMP in plants grown with various Pi concentrations were determined, and concentrations estimated for chloroplasts, the organelle containing threonine synthase and cystathionine γ-synthase. Calculations indicated that for growth at standard external Pi (0.4 millimolar) or above, if total OPH were uniformly distributed within the plants, activities of the two enzymes in question would be severely inhibited, and each would fall two orders of magnitude below the amount required to provide threonine (plus isoleucine) or methionine adequate for growth. If OPH were restricted to chloroplasts, these inhibitions would be much less severe, resulting in enzyme activities approaching the required physiological amounts. Evidence is presented that even up to 50 millimolar external Pi, this ion does not limit production of threonine or methionine sufficiently to retard growth, consistent with the postulated localization of OPH within chloroplasts.     

Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism

Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments in vitro, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of de novo DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.     

Adaptation of Lemna paucicostata to Sublethal Methionine Deprivation

During initial exposure to 40 nanomolar propargylglycine (PAG), Lemna paucicostata colonies undergo abnormal fragmentation and a lag in frond emergence, most severe at 24 to 48 hours. Thereafter, frond emergence resumes and the frond/colony ratio rises. Such `adapted' plants withstand subculture into the same concentration of PAG without fragmentation or decreases in frond emergence, and display enhanced tolerance to higher concentrations. Adaptation is not dependent upon outgrowth of a few preexisting especially tolerant plants. Exogenous methionine prevents these events and overcomes the PAG-induced lag in frond emergence even after it is underway. These changes in frond emergence are not reflected in the rates of protein and wet weight accumulation which decrease by about 25% during the first 24 hours and continue unchanged thereafter. Cystathionine γ-synthase activity rapidly decreases to 9% of control during the first 12 hours of exposure to 40 nanomolar PAG but thereafter climbs to 12% of control. Studies of the uptake and internal concentration of PAG during these events are reported.

Exposure to a combination of 36 micromolar lysine plus 3 micromolar threonine is an alternative means to bring about sublethal methionine deprivation. Thus exposed, Lemna undergoes an analogous sequence of effects on morphology and growth which are preventable by exogenous methionine and which lead to an adapted state. Cystathionine γ-synthase specific activity in plants adapted to 36 micromolar lysine plus 3 micromolar threonine is 1.8 times control. However, addition of PAG showed that under these conditions enzyme activity can be decreased to as little as 54% of control without affecting the growth rate. Together these results suggest that adaptation is related to methionine limitation and that the plants adjust, in part, by increasing the steady-state concentrations of cystathionine γ-synthase and other enzymes in the methionine pathway.

     

The transsulfuration pathway in tetrahymena pyriformis

Four enzymes necessary for the metabolism of methione by the transsulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosyl-homocysteinase (EC 3.3.1.1), cystathionine β-synthase (EC 4.2.1.22) and cystathionine γ-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer ³⁵S from [³⁵S] methionine to form [³⁵S] - cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, $\text{S-}\text{adenosylmethionine}$, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.     

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.     

Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.     

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.     

Analysis of Corynebacterium glutamicum methionine biosynthetic pathway: isolation and analysis of metB encoding cystathionine gamma-synthase.

The metB gene encoding cystathionine y-synthase, the second enzyme of methionine biosynthetic pathway, was isolated from a pSL109-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli metB mutant. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 1161 bp which encodes a protein with the molecular weight of 41,655 comprising of 386 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. Introduction of a plasmid carrying the cloned metB into the C. glutamicum resulted in a 10-fold increase in cystathionine gamma-synthase activities, demonstrating the identity of the cloned gene. The C. glutamicum metB mutant which was generated by the site-specific integration of the cloned DNA into its chromosome did not lose the ability to grow on glucose minimal medium lacking supplemental methionine. The growth rate of the mutant strain was also comparable to that of the parental strain. These data indicate that, in addition to the transsulfuration pathway, other methionine biosynthetic pathways may be present in C. glutamicum.     

Metabolism of 5′-methylthioadenosine in Aspergillus nidulans an alternative pathway for methionine synthesis via utilization of the nucleoside methylthio group

Experiments in which 5′-methylthioadenosine was used as a culture supplement for methionine-requiring mutants of Aspergillus nidulans with various enzymatic lesions indicated that the methylthio group derived from the nucleoside can be recycled to methionine. The results strongly suggest that methionine may be synthesized in the reaction catalyzed by homocysteine synthase (EC 4.2.99.10) in which O-acetylhomoserine is an acceptor of the methylthio group. The first step on the salvage pathway of the methylthio group is, in Aspergillus nidulans, phosphorolytic cleavage of 5′-methylthioadenosine to adenine and 5-methylthioribose 1-phosphate catalyzed by a specific phosphorylase.     

l-NAME has Opposite Effects on the Productions of S-adenosylhomocysteine and S-adenosylmethionine in V12-H-Ras and M-CR3B-Ras Pheochromocytoma Cells

Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. Formed homocysteine is either catabolized into cystathionine (transsulfuration pathway) by cystathionine β-synthase, or remethylated into methionine (remethylation pathway) by methionine synthase. To demonstrate the specificity of Ras-elicited effects on the activity of methyl cycles, wild-type pheochromocytoma PC12, mutant oncogenic rasH gene (MVR) expressing PC12 pheochromocytoma and normal c-rasH stably transfected M-CR3B cells were incubated with the N^ω-nitro-l-arginine methyl ester (l-NAME), and manumycin, (inhibitors of nitric oxide synthase and farnesyltransferase, respectively). We have found that l-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was decreased. We conclude that depletion of endogenous NO with l-NAME increased the production of SAH only in cells with mutated oncogenic RasH, possibly through enhancement of production of reactive oxygen species (ROS). Oxidative stress can increase cystathionine β-synthase activity that switches methyl cycles from remethylation into transsulfuration pathway to maintain the intracellular glutathione pool (essential for the redox-regulating capacity of cells) via an adaptive process.     

The Regulation of l-Methionine Synthesis and the Properties of Cystathionine γ-Synthase and β-Cystathionase in Corynebacterimn glutamicum

Cystathionine γ-synthase and β-cystathionase activities were found to be present in cell-free extract of Corynebacterium glutamicum. The reactions catalyzed by cystathionine γ-synthase and β-cystathionase were characterized with respect to Michaelis constant, pH optimum, incubation time and optimal enzyme concentration. Cystathionine γ-synthase was sensitive to the inhibition by S-adenosylmethionine. Formation of cystathionine γ-synthase and β-cystathionase was repressed by the addition of methionine to the growth medium although this repression appeared to be non-coordinate.

The regulation of methionine biosynthesis in C. glutamicum was discussed on the basis of these findings.     

Cystathionine γ-Lyase-deficient Mice Require Dietary Cysteine to Protect against Acute Lethal Myopathy and Oxidative Injury

Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine γ-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth^−/−) mice as an animal model of cystathioninemia/cystathioninuria. Cth^−/− mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth^−/− mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth^−/− hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth^−/− mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine β-synthase (Cbs^−/−). Because Cbs^−/− mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie''s method, which detects hypermethioninemia.