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This paper reports studies of two patients proven by a variety of studies to have mitochondrial depletion syndromes due to mutations in either their MPV17 or DGUOK genes. Each was initially investigated metabolically because of plasma methionine concentrations as high as 15-21-fold above the upper limit of the reference range, then found also to have plasma levels of S-adenosylmethionine (AdoMet) 4.4-8.6-fold above the upper limit of the reference range. Assays of S-adenosylhomocysteine, total homocysteine, cystathionine, sarcosine, and other relevant metabolites and studies of their gene encoding glycine N-methyltransferase produced evidence suggesting they had none of the known causes of elevated methionine with or without elevated AdoMet. Patient 1 grew slowly and intermittently, but was cognitively normal. At age 7 years he was found to have hepatocellular carcinoma, underwent a liver transplant and died of progressive liver and renal failure at age almost 9 years. Patient 2 had a clinical course typical of DGUOK deficiency and died at age 8 ½ months. Although each patient had liver abnormalities, evidence is presented that such abnormalities are very unlikely to explain their elevations of AdoMet or the extent of their hypermethioninemias. A working hypothesis is presented suggesting that with mitochondrial depletion the normal usage of AdoMet by mitochondria is impaired, AdoMet accumulates in the cytoplasm of affected cells poor in glycine N-methyltransferase activity, the accumulated AdoMet causes methionine to accumulate by inhibiting activity of methionine adenosyltransferase II, and that both AdoMet and methionine consequently leak abnormally into the plasma.

This review covers briefly the major conditions, genetic and non-genetic, sometimes leading to abnormally elevated methionine, with emphasis on recent developments. A major aim is to assist in the differential diagnosis of hypermethioninemia. The genetic conditions are: (1) Homocystinuria due to cystathionine β-synthase (CBS) deficiency. At least 150 different mutations in the CBS gene have been identified since this deficiency was established in 1964. Hypermethioninemia is due chiefly to remethylation of the accumulated homocysteine. (2) Deficient activity of methionine adenosyltransferases I and III (MAT I/III), the isoenzymes the catalytic subunit of which are encoded by MAT1A. Methionine accumulates because its conversion to S-adenosylmethionine (AdoMet) is impaired. (3) Glycine N-methyltrasferase (GNMT) deficiency. Disruption of a quantitatively major pathway for AdoMet disposal leads to AdoMet accumulation with secondary down-regulation of methionine flux into AdoMet. (4) S-adenosylhomocysteine (AdoHcy) hydrolase (AHCY) deficiency. Not being catabolized normally, AdoHcy accumulates and inhibits many AdoMet-dependent methyltransferases, producing accumulation of AdoMet and, thereby, hypermethioninemia. (5) Citrin deficiency, found chiefly in Asian countries. Lack of this mitochondrial aspartate-glutamate transporter may produce (usually transient) hypermethioninemia, the immediate cause of which remains uncertain. (6) Fumarylacetoacetate hydrolase (FAH) deficiency (tyrosinemia type I) may lead to hypermethioninemia secondary either to liver damage and/or to accumulation of fumarylacetoacetate, an inhibitor of the high K(m) MAT. Additional possible genetic causes of hypermethioninemia accompanied by elevations of plasma AdoMet include mitochondrial disorders (the specificity and frequency of which remain to be elucidated). Non-genetic conditions include: (a) Liver disease, which may cause hypermethioninemia, mild, or severe. (b) Low-birth-weight and/or prematurity which may cause transient hypermethioninemia. (c) Ingestion of relatively large amounts of methionine which, even in full-term, normal-birth-weight babies may cause hypermethioninemia.

A child with severe S-adenosylhomocysteine hydrolase (AHCY) deficiency (AHCY c.428A > G, p.Tyr143Cys ; c.982 T > G, p.Tyr328Asp) presented at 8 months of age with growth failure, microcephaly, global developmental delay, myopathy, hepatopathy, and factor VII deficiency. Plasma methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) were markedly elevated and the molar concentration ratio of AdoMet:AdoHcy, believed to regulate a myriad of methyltransferase reactions, was 15% of the control mean. Dietary therapy failed to normalize biochemical markers or alter the AdoMet to AdoHcy molar concentration ratio. At 40 months of age, the proband received a liver segment from a healthy, unrelated living donor. Mean AdoHcy decreased 96% and the AdoMet:AdoHcy concentration ratio improved from 0.52 ± 0.19 to 1.48 ± 0.79 mol:mol (control 4.10 ± 2.11 mol:mol). Blood methionine and AdoMet were normal and stable during 6 months of follow-up on an unrestricted diet. Average calculated tissue methyltransferase activity increased from 43 ± 26% to 60 ± 22%, accompanied by signs of increased transmethylation in vivo. Factor VII activity increased from 12% to 100%. During 6 postoperative months, head growth accelerated 4-fold and the patient made promising gains in gross motor, language, and social skills.

Studies were carried out to identify the cause of combined severe hypermethioninemia and moderate hyperhomocysteinemia in a cluster of 10 infants ascertained between 1999 and early 2001. Although several were thought initially to have cystathionine beta-synthase (CBS) deficiency and treated accordingly, CBS deficiency and other known genetic causes of hypermethioninemia were ruled out by assay of CBS activity in fibroblasts of four patients and by assays of plasma cystathionine and S-adenosylmethionine. Retrospective data on dietary methionine intakes and plasma concentrations of methionine and related metabolites established that the hypermethioninemia in nine of the 10 babies was related to ingestion of an infant protein hydrolysate formula, the methionine content of which had been increased from May 1998 to February 2001. The formula in question has now been reformulated and is no longer available. The 10th infant manifested similar metabolic abnormalities while receiving TPN containing excessive methionine. Brain MRI abnormalities indicative of cerebral edema, most marked in the cerebral cortex and posterior brainstem, occurred in two patients near times of extreme hypermethioninemia. Metabolic and MRI abnormalities resolved when the methionine intake decreased. A third infant had a normal MRI 1 day after the formula was changed. The possible relationship between extreme hypermethioninemia and cerebral edema is discussed and a working hypothesis offered to explain the relative sensitivity of the inferior colliculi, based upon the facts that this is the region most active in glucose utilization and that Na(+),K(+)-ATPase is inhibited by methionine and related metabolites.

Cystathionine beta-synthase (CBS) deficiency, the most common form of homocystinuria, is an autosomal recessive inborn error of homocysteine metabolism. Treatment of B6-nonresponsive patients centers on lowering homocysteine and its disulfide derivatives (tHcy) by adherence to a methionine-restricted diet. However, lifelong dietary control is difficult. Betaine supplementation is used extensively in CBS-deficient patients to lower plasma tHcy. With betaine therapy, methionine levels increase over baseline, but usually remain below 1,500 micromol/L, and these levels have not been associated with adverse affects. We report a child with B6-nonresponsive CBS deficiency and dietary noncompliance whose methionine levels reached 3,000 micromol/L on betaine, and who subsequently developed massive cerebral edema without evidence of thrombosis. We investigated the etiology by determining methionine and betaine metabolites in our patient, and several possible mechanisms for her unusual response to betaine are discussed. We conclude that the cerebral edema was most likely precipitated by the betaine therapy, although the exact mechanism is uncertain. This case cautions physicians to monitor methionine levels in CBS-deficient patients on betaine and to consider betaine as an adjunct, not an alternative, to dietary control.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields aMAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PPi. Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.

The concentrations of methionine and S-adenosylmethionine (AdoMet) in plasma and free choline and phospholipid-bound choline in both plasma and red blood cells from individuals with isolated hypermethioninemia have been measured. The only genetic abnormalities identified in these individuals have been inactivating mutations in MAT1A, the gene that encodes the subunit of the isozymes of methionine adenosyltransferase (MAT), MAT I, and MAT III, expressed only in adult liver. These measurements were performed to learn more about AdoMet metabolism and to test the working hypotheses that inadequate delivery of AdoMet, or of choline or a choline derivative, from liver to brain might be a cause of the neurologic disease often found in humans with the most severe losses of MAT I/III activity. In striking contrast to the elevations of plasma AdoMet reported in control humans with hypermethioninemia resulting from methionine loading, plasma AdoMet levels were generally below the mean reference value in the MAT I/III-deficient hypermethioninemic patients. This is interpreted as a result of subnormal formation of AdoMet in liver due to the deficient activity of MAT I/III and resultant lower-than-normal delivery of AdoMet from liver to plasma. A low plasma AdoMet concentration in the presence of an elevated methionine provides a useful diagnostic tool that pinpoints the cause of a case of hypermethioninemia as defective MAT I/III activity. Plasma-free choline concentrations were also generally somewhat below normal in the hypermethioninemic patients. However, neither plasma AdoMet nor plasma choline concentrations were strikingly lower in MAT I/III-deficient individuals with neurologic abnormalities than in those without. These results thus fail to provide support for the working hypotheses in question.

Background There is not much information on established standard therapy for patients with severe methionine adenosyltransferase (MAT) I/III deficiency. Case presentation We report a boy with MAT I/III deficiency, in whom plasma methionine and total homocysteine, and urinary homocystine were elevated. Molecular genetic studies showed him to have novel compound heterozygous mutations of the MAT1A gene: c.191T>A (p.M64K) and c.589delC (p.P197LfsX26). A low methionine milk diet was started at 31 days of age, and during continuing dietary methionine restriction plasma methionine levels have been maintained at less than 750 μmol/L. He is now 5 years old, and has had entirely normal physical growth and psychomotor development. Conclusions Although some severely MAT I/III deficient patients have developed neurologic abnormalities, we report here the case of a boy who has remained neurologically and otherwise normal for 5 years during methionine restriction, suggesting that perhaps such management, started in early infancy, may help prevent neurological complications.

S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status.

We describe a family illustrating the diagnostic difficulties occurring when pyridoxine-responsive cystathionine beta-synthase (CBS) deficiency presents with thrombotic disease without associated ocular, skeletal, or CNS abnormalities, a situation increasingly recognized. This family had several thromboembolic episodes in two generations with apparently inconstant elevations of plasma total homocysteine (tHcy). When taking (sometimes even low amounts) of pyridoxine, the affected family members had low-normal tHcy and normal values for cystathionine, methionine, and cysteine. Withdrawal of vitamin therapy was necessary before lower cystathionine, elevated methionine, and decreased cysteine became apparent, a pattern suggestive of CBS deficiency, leading to the finding that the affected members were each compound heterozygotes for CBS p.G307S and p.P49L. To assist more accurate diagnosis of adults presenting with thrombophilia found to have elevated tHcy, the patterns of methionine-related metabolites in CBS-deficient patients are compared in this article to those in patients with homocysteine remethylation defects, including inborn errors of folate or cobalamin metabolism, and untreated severe cobalamin or folate deficiency. Usually serum cystathionine is low in subjects with CBS deficiency and elevated in those with remethylation defects. S-Adenosylmethionine and S-adenosylhomocysteine are often markedly elevated in CBS deficiency when tHcy is above 100 umol/L. We conclude that there are likely other undiagnosed, highly B6-responsive adult patients with CBS deficiency, and that additional testing of cystathionine, total cysteine, methionine, and S-adenosylmethionine will be helpful in diagnosing them correctly and distinguishing CBS deficiency from remethylation defects.

Methionine adenosyltransferases (MAT's) are central enzymes in living organisms that have been conserved with a high degree of homology among species. In the liver, MAT I and III, tetrameric and dimeric isoforms of the same catalytic subunit encoded by the gene MAT1A, account for the predominant portion of total body synthesis of S-adenosylmethionine (SAM), a versatile sulfonium ion-containing molecule involved in a variety of vital metabolic reactions and in the control of hepatocyte proliferation and differentiation. During the past 15 years 28 MAT1A mutations have been described in patients with elevated plasma methionines, total homocysteines at most only moderately elevated, and normal levels of tyrosine and other aminoacids. In this study we describe functional analyses that determine the MAT and tripolyphosphatase (PPPase) activities of 18 MAT1A variants, six of them novel, and none of them previously assayed for activity. With the exception of G69S and Y92H, all recombinant proteins showed impairment (usually severe) of MAT activity. Tripolyphosphate (PPPi) hydrolysis was decreased only in some mutant proteins but, when it was decreased MAT activity was always also impaired.

Over a four-year period, we collected clinical and biochemical data from five Amish children who were homozygous for missense mutations in 5,10-methylenetetrahydrofolate reductase (MTHFR c.1129C>T). The four oldest patients had irreversible brain damage prior to diagnosis. The youngest child, diagnosed and started on betaine therapy as a newborn, is healthy at her present age of three years. We compared biochemical data among four groups: 16 control subjects, eight heterozygous parents, and five affected children (for the latter group, both before and during treatment with betaine anhydrous). Plasma amino acid concentrations were used to estimate changes in cerebral methionine uptake resulting from betaine therapy. In all affected children, treatment with betaine (534+/-222 mg/kg/day) increased plasma S-adenosylmethionine, improved markers of tissue methyltransferase activity, and resulted in a threefold increase of calculated brain methionine uptake. Betaine therapy did not normalize plasma total homocysteine, nor did it correct cerebral 5-methyltetrahydrofolate deficiency. We conclude that when the 5-methyltetrahydrofolate content of brain tissue is low, dietary betaine sufficient to increase brain methionine uptake may compensate for impaired cerebral methionine recycling. To effectively support the metabolic requirements of rapid brain growth, a large dose of betaine should be started early in life.

Various questions have been raised about labile methyl balance and total transmethylation fluxes, and further discussion has been encouraged. This report reviews and discusses some of the relevant evidence now available. The fact that, if needed, labile methyl balance is maintained by methylneogenesis appears to be established, but several aspects of transmethylation remain uncertain: definitive measurements of the rate of total transmethylation in humans of both sexes on various diets and at various ages; the extent to which synthesis of phosphatidylcholine has been underestimated; and the relative contributions of the 2 pathways for the formation of sarcosine (ie, N-methylglycine). The available evidence indicates that the quantitatively most important pathways for S-adenosylmethionine-dependent transmethylation in mammals are the syntheses of creatine by guanidinoacetate methyltransferase, of phosphatidylcholine by phosphatidylethanolamine methyltransferase, and of sarcosine by glycine N-methyltransferase. Data presented in this report show that S-adenosylmethionine and methionine accumulate abnormally in the plasma of humans with glycine N-methyltransferase deficiency but not of those with guanidinoacetate N-methyltransferase deficiency or in the plasma or livers of mice devoid of phosphatidylethanolamine N-methyltransferase activity. The absence of such accumulations in the latter 2 conditions may be due to removal of S-adenosylmethionine by synthesis of sarcosine. Steps that may help clarify the remaining issues include the determination of the relative rates of synthesis of sarcosine, creatine, and phosphatidylcholine by rapid measurement of the rates of radiolabel incorporation into these compounds from L-[methyl-3H]methionine administered intraportally to an experimental animal; clarification of the intracellular hepatic isotope enrichment value during stable-isotope infusion studies to enhance the certainty of methyl flux estimates during such studies; and definitive measurement of the dietary betaine intake from various diets.

We report studies of a Croatian boy, a proven case of human S -adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 μM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5–15.9 μM. In plasma, S -adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was ≈3% of control in liver and was 5–10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patient's cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.

Abnormal elevation of plasma methionine may result from several different genetic abnormalities, including deficiency of cystathionine [beta ]-synthase (CBS) or of the isoenzymes of methionine adenosyltransferase (MAT) I and III expressed solely in nonfetal liver (MAT I/III deficiency). Classically, these conditions have been distinguished most readily by the presence or absence, respectively, of elevated plasma free homocystine, detected by amino acid chromatography in the former condition, but absent in the latter. During the present work, we have assayed methionine, S-adenosylmethionine, S-adenosylhomocysteine, total homocysteine (tHcy), cystathionine, N-methylglycine (sarcosine), and total cysteine (tCys) in groups of both MAT I/III- and CBS-deficient patients to provide more evidence as to their metabolite patterns. Unexpectedly, we found that MAT I/III-deficient patients with the most markedly elevated levels of plasma methionine also had elevations of plasma tHcy and often mildly elevated plasma cystathionine. Evidence is presented that methionine does not inhibit cystathionine [beta ]-synthase, but does inhibit cystathionine gamma-lyase. Mechanisms that may possibly underlie the elevations of plasma tHcy and cystathionine are discussed. The combination of elevated methionine plus elevated tHcy may lead to the mistaken conclusion that an MAT I/III-deficient patient is instead CBS-deficient. Less than optimal management is then a real possibility. Measurements of plasma cystathionine, S-adenosylmethionine, and sarcosine should permit ready distinction between the 2 conditions in question, as well as be useful in several other situations involving abnormalities of methionine and/or homocysteine derivatives.

Cystathionine beta-synthase (CBS) deficiency, the most common form of homocystinuria, is an autosomal recessive inborn error of homocysteine metabolism. Treatment of B6-nonresponsive patients centers on lowering homocysteine and its disulfide derivatives (tHcy) by adherence to a methionine-restricted diet. However, lifelong dietary control is difficult. Betaine supplementation is used extensively in CBS-deficient patients to lower plasma tHcy. With betaine therapy, methionine levels increase over baseline, but usually remain below 1,500 micromol/L, and these levels have not been associated with adverse affects. We report a child with B6-nonresponsive CBS deficiency and dietary noncompliance whose methionine levels reached 3,000 micromol/L on betaine, and who subsequently developed massive cerebral edema without evidence of thrombosis. We investigated the etiology by determining methionine and betaine metabolites in our patient, and several possible mechanisms for her unusual response to betaine are discussed. We conclude that the cerebral edema was most likely precipitated by the betaine therapy, although the exact mechanism is uncertain. This case cautions physicians to monitor methionine levels in CBS-deficient patients on betaine and to consider betaine as an adjunct, not an alternative, to dietary control.

A search was made for compounds that would inhibit methionine biosynthesis in Lemna paucicostata Hegelm. 6746. dl-Propargylglycine (0.15 micromolar) produced growth inhibition and morphological changes which were prevented by exogenous methionine. Also, dl-propargylglycine inhibits cystathionine gamma-synthase activity. l-Aminoethoxyvinylglycine (0.05 micromolar) produced growth inhibition and morphological changes partially preventable by exogenous methionine. l-Aminoethoxyvinylglycine impairs the cleavage of cystathionine to homocysteine. Lysine and threonine, at concentrations which individually had little effect on growth or morphology of Lemna, together produced growth inhibition and morphological changes preventable by exogenous methionine. The resulting metabolic block prevented conversion of cysteine to cystathionine, presumably secondary to depletion of the supply of O-phosphohomoserine.Inhibition of Lemna growth resulted when the molybdate:sulfate ratio in the medium was increased to 20:1 or more. Such inhibition was prevented by lowering this ratio to 0.3 or less. A non-steady-state experiment (molybdate:sulfate, 20:1) showed that molybdate inhibited sulfate uptake, but it provided no evidence of a further impairment in the organification of sulfate. Molybdate-induced growth inhibition of Lemna was prevented by cystine but not by cystathionine or methionine. Cystathionine is not converted by Lemna to cysteine rapidly enough to sustain growth.

Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 μm, was labeled to radioisotopic equilibrium with 35 SO 4 2− . Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. γ-Glutamylcyst(e)ine, erythro - and threo -thiothreonine, and S-methylcysteine were not detected. No volatile 35 S compounds were formed during plant growth at 1,000 μm sulfate, nor were significant amounts of 35 S compounds excreted into the medium. The amount of each component present in colonies grown over the 3,000-fold range of medium sulfate was relatively constant except for inorganic sulfate. This increased about 30-fold from the lowest to the highest medium sulfate concentration. The total soluble sulfur amino acids increased about 1.5- to 2-fold, due primarily to an increased amount of glutathione. Protein cyst(e)ine and protein methionine were the major organic sulfur compounds in Lemna , and the amounts of these compounds remained virtually constant despite the variation in external sulfate concentration. Procedures for the analysis of S-adenosylmethionine, S-methylmethionine sulfonium, and S-adenosylhomocysteine are presented.

Extracts of phylogenetically diverse plans were surveyed for their ability to synthesize the following homoserine esters which are potential precursors for methionine and threonine synthesis in green plants: O -acetyl-, O -oxalyl-, O -succinyl-, O -malonyl-, and O -phosphohomoserine. Synthesis of O -acylhomoserine esters was detected only in Pisum sativum L. and Lathyrus sativus L. Extracts of P. sativum , a plant known to accumulate O -acetylhomoserine, catalyzed the specific synthesis of this ester from homoserine and acetyl-CoA. Extracts of L. sativus , a plant known to accumulate O -oxalylhomoserine, catalyzed the specific synthesis of this ester from homoserine and oxalyl-CoA. None of the other plants surveyed, including representatives of the green algae, horsetails, gymnosperms, and angiosperms, catalyzed the synthesis of any of the O -acylhomoserine esters studied. In contrast, synthesis of O -phosphohomoserine by the reaction catalyzed by homoserine kinase was demonstrated in extracts of all plants examined, including the two exceptional legumes. These results suggest that, among the five homoserine esters studied, O -phosphohomoserine is the major activated homoserine derivative in plants. Direct confirmation of the dominant physiological role of O -phosphohomoserine in the synthesis of cystathionine in the transsulfuration pathway of methionine biosynthesis in plants has recently been provided (Datko, A. H., Giovanelli, J., and Mudd, S. H. 1974. J. Biol. Chem. 249: 1139-1155).

The pathway for synthesis of phosphatidylcholine, the dominant methyl-containing end product formed by Lemna paucicostata, has been investigated. Methyl groups originating in methionine are rapidly utilized by intact plants to methylate phosphoethanolamine successively to the mono-, di-, and tri-methyl (i.e. phosphocholine) phosphoethanolamine derivatives. With continued labeling, radioactivity initially builds up in these compounds, then passes on, accumulating chiefly in phosphatidylcholine (34% of the total radioactivity taken up by plants labeled to isotopic equilibrium with l-[(14)CH(3)]methionine), and in lesser amounts in soluble choline (6%). Radioactivity was detected in mono- and dimethyl derivatives of free ethanolamine or phosphatidylethanolamine only in trace amounts. Pulse-chase experiments with [(14)CH(3)]choline and [(3)H] ethanolamine confirmed that phosphoethanolamine is rapidly methylated and that phosphocholine is converted to phosphatidylcholine. Initial rates indicate that methylation of phosphoethanolamine predominates over methylation of either phosphatidylethanolamine or free ethanolamine at least 99:1. Although more studies are needed, it is suggested this pathway may well turn out to account for most phosphatidylcholine synthesis in higher plants. Phosphomethylethanolamine and phosphodimethylethanolamine are present in low quantities during steady-state growth (18% and 6%, respectively, of the amount of phosphocholine). Radioactivity was not detected in CDP-choline, probably due to the low steady-state concentration of this nucleotide.

To provide information upon the ways in which Lemna paucicostata uses the methyl group of methionine, plants were grown for various periods (from 1 minute to 6.8 days) in the presence of a tracer dose of radioactive methyl-labeled methionine. Protein methionine accounted for approximately 19% of the accumulated methyl moieties; other methylated products, about 81%. The latter group included (percent of total methyl in parentheses): methylated ethanolamine derivatives (46%); methyl esters of the pellet (chiefly, or solely, pectin methyl esters) (15%); chlorophyll methyl esters (8%); unidentified neutral lipids (6%); nucleic acid derivatives (2-5%); methylated basic amino acids (2%). No other major methylated compounds were observed in any plant fraction. Available evidence suggests that little, if any, oxidation of the methyl group of methionine, directly or indirectly, occurs in Lemna. Our results indicate that S-methyl-methionine sulfonium is formed relatively rapidly, but does not accumulate at a commensurate rate, probably being reconverted to methionine. To our knowledge, this is the first time a reasonably complete accounting of the metabolic fate of methionine methyl has been obtained for any plant. The extent to which the results with Lemna may be representative of the situation for other higher plants is discussed.

The methylation steps in the biosynthesis of phosphatidylcholine by tissue culture preparations of carrot (Daucus carota L.) and soybean (Glycine max), and by soybean leaf discs, have been studied. Preparations were incubated with tracer concentrations of l-[(3)H(3)C]methionine and the kinetics of appearance of radioactivity in phosphomethylethanolamine, phosphodimethylethanolamine, phosphocholine, phosphatidylmethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, methylethanolamine, dimethylethanolamine, and choline followed at short incubation times. With soybean (tissue culture or leaves), an initial methylation utilizes phosphoethanolamine as substrate, forming phosphomethylethanolamine. The latter is converted to phosphatidylmethylethanolamine, which is successively methylated to phosphatidyldimethyethanolamine and to phosphatidylcholine. With carrot, again, an initial methylation is of phosphoethanolamine. Subsequent methylations occur at both the phospho-base and phosphatidyl-base levels. Both of these patterns differ qualitatively from that previously demonstrated in Lemna (SH Mudd, AH Datko 1986 Plant Physiol 82: 126-135) in which all three methylations occur at the phospho-base level. For soybean and carrot, some added contribution from initial methylation of phosphatidylethanolamine has not been excluded. These results, together with those from similar experiments carried out with water-stressed barley leaves (WD Hitz, D Rhodes, AD Hanson 1981 Plant Physiol 68: 814-822) and salinized sugarbeet leaves (AD Hanson, D Rhodes 1983 Plant Physiol 71: 692-700) suggest that in higher plants some, perhaps all, phosphatidylcholine synthesis occurs via a common committing step (conversion of phosphoethanolamine to phosphomethylethanolamine) followed by a methylation pattern which differs from plant to plant.

Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine gamma-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 gamma-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.

Individual rates of metabolism of the sulfur, methyl, and 4-carbon moieties of methionine were estimated in Lemna paucicostata Hegelm. 6746 growing under standard conditions, and used to quantitate pathways of methionine metabolism. Synthesis of S-adenosylmethionine (AdoMet) is the major pathway for methionine metabolism, with over 4 times as much methionine metabolized by this route as accumulates in protein. More than 90% of AdoMet is used for transmethylation. Methyl groups of choline, phosphatidylcholine, and phosphorylcholine are major end products of this pathway. Flux through methylthio recycling is about one-third the amount of methionine accumulating in protein. Spermidine synthesis accounts for at least 60% of the flux through methylthio recycling. The results obtained here, together with those reported for methionine-supplemented plants (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839), indicate that methionine supplementation reduced methylneogenesis by no more than the small amount expected from the reduced entry of sulfate sulfur into methionine (Giovanelli, Mudd, Datko, 1985 Plant Physiol 77: 450-455). Methionine supplementation had no significant effect on transmethylation or methylthio recycling. The combined data provide the first comprehensive estimates of the quantitative relationships of major pathways for methionine metabolism and their control in plants.

The clinical and biochemical findings in two brothers each of whom has homocystinuria and methylmalonic aciduria are described. Studies of fibroblasts grown from skin biopsies of these patients demonstrated that their homocystinuria is due to decreased activity of N5-methyltetra-hydrofolate methyltransferase, rather than due to decreased activity of cystathionine synthase. These siblings are the second and third reported cases of homocystinuria due to a defect in homocysteine methylation. Both the means and importance of differentiating homocystinuria due to cystathionine synthase deficiency from that due to defects in methylation of homocysteine are discussed. The cause of the methylmalonic aciduria has not been finally established, although the data are compatible with a partial decrease in activity of methylmalonyl-CoA isomerase. Together, the abnormalities in these brothers may tentatively be ascribed to a defect in the uptake or early metabolism of B12, the condition most likely being inherited as an autosomal recessive trait.

An infant is described who died at seven and one half weeks of age and whose clinical manifestations included poor feeding, hematemesis, melena, normocytic anemia and failure to gain weight. He was found to accumulate abnormally large amounts of cystathionine, homocystine and homocysteine-cysteine mixed disulfide and to have low concentrations of methionine in blood and urine. In addition he manifested a marked methylmalonic aciduria that did not respond to the intramuscular administration of vitamin B, (cyanocobalamin). Enzymatic analyses of liver, kidney and brain obtained at autopsy revealed specifically deficient activity in the B,-dependent enzyme, N,-methyltetrahydrofolate-homocysteine methyltransferase. Studies of fibroblasts grown from skin obtained ante mortem showed that the activity of the B--dependent enzyme, methylmalenyl-CoA (coenzyme A) isomerase, also was deficient, but this activity could be restored by growing the cells in medium containing excess hydroxocobalamin. Analysis of liver and kidney revealed that the concentration of deoxyadenosyl cobalamin (coenzyme-B,) was abnormally low whereas the concentration of total B, was high in serum and normal in liver. Apparently this infant could not adequately metabolize B, and as a result failed to accumulate coenzymatically-active derivatives of vitamin B, in normal amounts. It is suggested that therapeutic benefit may derive from the administration of forms of 6, that could circumvent the metabolic block, by giving substances that could stimulate the non-B, -dependent methionine-synthesizing system or by the administration of methionine in large doses.

1. Hepatic levels of cystathionine synthase and methionine-activating enzyme are significantly lower in rats fed a diet low in methionine and supplemented with cystine than in rats growing at the same rate while maintained on a diet adequate in methionine, with or without cystine supplementation. Cystathionase levels are also decreased, but to a smaller extent. Betaine-homocysteine methyltransferase is not affected. 2. The enzymatic activities which are lowered are restored toward normal by injections of l-methionine or l-homocysteine. 3. Methionine-activating enzyme and cystathionine synthase are inhibited in vitro by l-cystine. However, the decreased enzyme levels in the livers of rats fed the low methionine, cystine-supplemented diet cannot be attributed to either a dissociable inhibitor or cystine binding by the enzyme proteins. It seems likely that the cystine effect represents repression of enzyme synthesis. 4. The physiological meaning of these changes in enzymatic activity is briefly discussed. The changes are such that they may well explain the known "methionine-sparing" effect of cystine. 5. A possible application of these findings to the treatment of patients with homocystinuria due to cystathionine synthase deficiency is mentioned.

Studies are reported of fibroblasts cultured from the skin of a patient with abnormalities of sulfur amino acid and of methylmalonic acid metabolism. These cells are unable to grow as do normal cells if methionine in the medium is replaced by homocystine. Addition of hydroxy-B12, 1 μg/ml, to the medium allowed the cells of the patient to grow slowly. Extracts of the cells contained a normal concentration of cystathionine synthase, but an abnormally low concentration of holoenzyme for N5-methyltetrahydrofolate methyltransferase activity. N5-Methyltetrahydrofolate methyltransferase apoenzyme was present, as indicated by assay in the presence of appropriate B12-containing compounds. Growth of control cells in media containing hydroxy-B12, 1 μg/ml, led to a several-fold increase in total methyltransferase activity and a decrease of dependence of activity upon in vitro supplementation with B12-containing compounds. Growth of the patient's cells under these conditions led to formation of a small, but detectable, amount of holoenzyme. However, the major part of the methyltransferase activity in extracts of his cells grown on hydroxy-B12 remained dependent upon in vitro addition of a B12-containing compound. The patient's cells converted propionate-1-14C to 14CO2 at a slow rate compared to control cells. During incubation with propionate-1-14C his cells accumulated abnormally great amounts of methylmalonate-14C but failed to form the usual amounts of radioactive succinate, lactate, glutamate, or aspartate. Growth of the patient's cells with supplemental hydroxy-B12 in the medium partially restored the rate at which these cells converted propionate-1-14C to 14CO2. The specificity of this restorative effect as well as the effects of variation in concentration and time of exposure to hydroxy-B12 are reported. All the available data are consistent with the hypothesis that cells from the patient are deficient in their ability to accumulate coenzymatically active derivatives of B12 with resultant deficits in the activities of N5-methyltetrahydrofolate methyltransferase and methylmalonyl-CoA isomerase, the two enzymes known in mammals to depend upon B12 derivatives.

Summary Methionine adenosyltransferase (MAT) I/III deficiency, caused by mutations in the MAT1A gene, is characterized by persistent hypermethioninemia without elevated homocysteine or tyrosine. Clinical manifestations are variable and poorly understood, although a number of individuals with homozygous null mutations in MAT1A have neurological problems, including brain demyelination. We analyzed MAT1A in seven hypermethioninemic individuals, to provide insight into the relationship between genotype and phenotype. We identified six novel mutations and demonstrated that mutations resulting in high plasma methionines may signal clinical difficulties. Two patients—a compound heterozygote for truncating and severely inactivating missense mutations and a homozygote for an aberrant splicing MAT1A mutation—have plasma methionine in the 1,226–1,870 μM range (normal 5–35 μM) and manifest abnormalities of the brain gray matter or signs of brain demyelination. Another compound heterozygote for truncating and inactivating missense mutations has 770–1,240 μM plasma methionine and mild cognitive impairment. Four individuals carrying either two inactivating missense mutations or the single-allelic R264H mutation have 105–467 μM plasma methionine and are clinically unaffected. Our data underscore the necessity of further studies to firmly establish the relationship between genotypes in MAT I/III deficiency and clinical phenotypes, to elucidate the molecular bases of variability in manifestations of MAT1A mutations.

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.

Homocystinuria and homocystinemia without hypermthioninemia, but with reccurent episodes of folate responseive schizophrenic-like behavior, was documented in a mildly retarded adolescent girl who lacked the habitus associated with cystathionine synthase deficiency. Enzymes involved in homocysteine-methionine metabolism were demonstrated to be normal. A defect in the ability to reducte N-5-10--methylenetetrahydrofolate to 5-methyltetrahydrofolate was demonstrated. Methylenetetrahydrofolate reductase was 18 per cent of control values. Methyltetrahydrofolate is used for the methylation of homocysteine to methionine, and a deficiency of this compound could explain the homocystinemia and homocystinuria.

Administration of methionine to growing Lemna had essentially no effect on accumulation of sulfate sulfur in protein cysteine, but decreased accumulation into cystathionine and its products (homocysteine, methionine, S-methylmethioninesulfonium salt, S-adenosylmethionine, and S-adenosylhomocysteine) to as low as 21% that of control plants, suggesting that methionine regulates its own de novo synthesis at cystathionine synthesis. Methionine caused only a slight reduction (to 80% that of control plants) in the accumulation of sucrose carbon into the 4-carbon moieties of cystathionine and products. This observation was puzzling since cystathionine synthesis proceeds by incorporation of equivalent amounts of sulfur (from cysteine) and 4-carbon moieties (from O-phosphohomoserine). The apparent inconsistency was resolved by the demonstration in Lemna (Giovanelli, Datko, Mudd, Thompson 1983 Plant Physiol 71: 319-326) that de novo synthesis of the methionine 4-carbon moiety occurs not only via the established transsulfuration route from O-phosphohomoserine, but also via the ribose moiety of 5'-methylthioadenosine. It is now clear that the more accurate assessment of the flux of sulfur (and 4-carbon moieties) through transsulfuration is provided by the amount of (35)S from (35)SO(4) (2-) that accumulates in cystathionine and its products, rather than by the corresponding measurements with (14)C. These studies therefore unequivocally demonstrate in higher plants that methionine does indeed feedback regulate it own de novo synthesis in vivo, and that cystathionine synthesis is a locus for this regulation.

Three circumstances prompted us to reexamine the relationship between abnormal cystathionine accumulation and possible homocystinuria resulting from this condition: (a) discovery of an infant girl with apparently alternating massive cystathioninuria and homocystinuria; (b) the presence of homocystinuria in some, but not all, previously reported cases of cystathioninuria probably due to γ-cystathionine deficiency; and (c) the recent demonstration that mammalian cystathionine s-synthase can cleave cystathionine to homocysteine. The following conclusions were reached: (a) Homocystine may arise as a result of bacteria, contamination of a urine sample initially containing cystathionine, but not homocystine. (b) After a methionine load, a cystathioninuric patient may excrete readily detected amounts of homocystine. (c) However, homocystinuria is not a necessary concomitant of even massive cystathioninuria. These findings and some of their implications are briefly discussed.

Normal young adult male and female subjects were maintained on fixed dietary regimens which were either essentially normal or were semisynthetic and curtailed in methionine and choline intakes and virtually free of cystine. The subjects maintained stable weights and remained in positive nitrogen balance or within the zone of sulfur equilibrium. Choline intakes were calculated, and urinary excretions of creatinine, creatine, and sacrosine were measured. Creatinine excretions of male subjects on essentially normal diets outweighed the total intakes of labile methyl groups. Taking into account the excretions of additional methylated compounds, as judged from published values, it appears that methyl neogenesis must normally play a role in both males and females. When labile methyl intake is curtailed, de novo formation of methyl groups is quantitatively more significant than ingestion of preformed methyl moieties. On the normal diets used in these experiments, the average homocysteinyl moiety in males cycled between methionine and homocysteine at least 1.9 times before being converted to cystathionine. For females, the average number of cycles was at least 1.5. When labile methyl intake was curtailed, the average number of cycles rose to 3.9 for males and 3.0 for females under the conditions employed.

Publisher Summary This chapter discusses sulfur amino acids in plants. Most of the inorganic sulfate assimilated and reduced by plants appears ultimately in cysteine and methionine. These amino acids contain about 90% of the total sulfur in most plants. Nearly all of the cysteine and methionine is in protein. The chapter highlights the typical dominance of protein cysteine and protein methionine in the total organic sulfur. In the nonprotein fraction, reduced glutathione (GSH) is ubiquitous and is commonly a major constituent. The soluble fraction of plants also includes a variety of other sulfur-containing compounds that are normally present in relatively small amounts: (1) intermediates on the route to protein cysteine and protein methionine, such as cysteine, cystathionine, homocysteine, and methionine and (2) compounds involved in methyl transfer reactions and polyamine synthesis such as AdoMet, AdoHcy, and 5'-methyl-thioadenosine. Cysteine is the precursor of protein cysteine, GSH, and the sulfur moiety of methionine and is, therefore, the major portal for organic reduced sulfur. Serine is the source of the C3 moiety of cysteine in plants.

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 gamma-synthase, establishing the separate identity of these enzymes. Compared to cystathionine gamma-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.No evidence was obtained for feedback inhibition, repression, or covalent modification of TS by threonine and/or isoleucine.

The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the path-way for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: serine --ethanolamine --phosphoethanolamine --phosphatidylethanolamine. A possible alternative is: serine --phosphatidylserine --phosphatidylethanolamine --ethanolamine --phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO(2) from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives.

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 gamma-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 gamma-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 gamma-synthase and other enzymes in the methionine pathway.

Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O -phosphohomoserine-dependent cystathionine γ-synthase, O -phosphohomoserine sulfhydrylase, and O -acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine γ-synthase, it was observed that (a) adding external methionine (2 μm) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 μml-aminoethoxyvinylglycine or with 36 μm lysine plus 4 μm threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S -adenosylmethionine, or S -methylmethionine sulfonium to the assay mixture. Parallel studies of O -phosphohomoserine sulfhydrylase and O -acetylserine sulfhydrylase showed that O -phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine γ-synthase activity, whereas O -acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O -acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O -acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O -phosphohomoserine sulfhydrylase. The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine γ-synthase in Lemna , (b) O -phosphohomoserine sulfhydrylase and cystathionine γ-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O -acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of O -acetylserine sulfhydrylase provides an explanation in molecular terms for transsulfuration, and not direct sulfhydration, being the dominant pathway for homocysteine biosynthesis.

The results of studies of SO 4 2− uptake by Lemna paucicostata are most simply interpreted by the hypothesis that at least two components are involved, one saturating and one linear, `nonsaturating.9 The saturating component has a low K m and high specificity for SO 4 2− . Uptake by the nonsaturating component is less affected by pH and temperature than is that of the saturating system. SO 4 2− efflux is not quantitatively important in Lemna under standard conditions (20 micromolar SO 4 2− ) (Datko AH, SH Mudd 1980 Plant Physiol 65: 906-912). 55% of newly taken up 35 SO 4 2− enters a slowly turning over compartment (vacuole?); 45% remains in a compartment (cytoplasm?) in which it is rapidly metabolized to organic compounds. Growth in increased concentrations of SO 4 2− or cystine, but not methionine, down-regulates the saturating, but not the nonsaturating, system. Growth in limiting SO 4 2− up-regulates the saturating system. Overall, a 500-fold change was observed. Reciprocal inhibition experiments demonstrated that molybdate and SO 4 2− are taken up by a common mechanism, but growth in molybdate failed to up-regulate SO 4 2− uptake. Regulation by growth in SO 4 2− or cystine did not markedly affect uptake of phosphate or of several organic compounds. The saturating system contributes 99% of SO 4 2− uptake under standard conditions, providing sufficient SO 4 2− so it is not limiting. In nature the same system likely contributes at least 65 to 70%.

Synthesis by plants of homocysteine, the immediate precursor of methionine, is a key reaction in biology (Allaway, 1970). This is so because non-ruminant animals require a dietary source of homocysteine, which is normally provided in the form of methionine. Animals metabolize these sulfur amino acids eventually to inorganic sulfate. Plants complete the cycle of sulfur by reductive assimilation of inorganic sulfate to methionine (and cysteine) (Siegel, 1975), and are thus the ultimate source of methionine in most animal diets.

The steady state concentrations of S-containing compounds formed in Lemna paucicostata Hegelm. 6746 in response to variations in source and concentrations of sulfur were measured. Neither growth rates nor protein accumulation were markedly affected by the various growth conditions. Ignoring complications due to possible compartmentation, the results are consistent with internal pools of both SO(4) (2-) and cyst(e)ine (or products of their metabolism), but not methionine, being effectors of regulation of high affinity SO(4) (2-) uptake. As SO(4) (2-) in the growth medium was increased to 10 mm, down-regulation of high affinity SO(4) (2-) uptake was more than compensated for by unregulated uptake via the "non-saturating" uptake system. Tissue inorganic SO(4) (2-) accumulated but formation of reduced sulfur remained constant. Some conversion of l-cystine sulfur to SO(4) (2-) occurred. Presence of l-cystine in the medium (a) down-regulated high affinity SO(4) (2-) uptake and (b) decreased the rate of SO(4) (2-) organification. The net results were decreased (7 mum l-cystine) or normal (14 mum l-cystine) total tissue SO(4) (2-) and dose-dependent accumulation of soluble cyst(e)ine and glutathione, but not of soluble methionine. l-Methionine was not metabolized to cyst(e)ine or its products. Presence of l-methionine in the medium led to increased total tissue sulfur, accounted for almost wholly by manyfold increases in soluble methionine, AdoMet, and S-methylmethionine sulfonium. Soluble cyst(e)ine increased slightly.

Summary When duckweed was grown in the presence of [35S, U-14C]-methionine the ratio of [14C]-4-carbon moiety relative to 35S in protein methionine was less than in the administered methionine. This decrease demonstrates a preferential recycling of the sulfur moiety relative to the 4-carbon moiety of methionine. This pathway probably plays a hitherto unsuspected general role in plants, such as in polyamine synthesis. The ratio of [14C]-methyl relative to 35S of protein methionine was also less than in the administered methionine. This decrease is tentatively attributed to a rapid utilization of the methyl moiety of methionine for transmethylation, accompanied by recycling of homocysteine to methionine. Transmethylation was estimated to be the dominant route for methionine metabolism.

Little, if any, regulation of threonine synthesis was observed in Lemna paucicostata Hegelm. 6746 supplemented with concentrations of threonine and/or isoleucine that allow for uptake of these amino acids in amounts sufficient for total plant requirements, and that increase tissue concentrations of soluble threonine manyfold. High tissue concentrations of soluble threonine generated endogenously in isoleucine-supplemented plants were no more effective in regulation than a similar concentration of threonine accumulated from the medium. These studies exclude also major regulation of threonine biosynthesis by bivalent repression by threonine plus isoleucine. Isoleucine biosynthesis was severely inhibited by supplementation with isoleucine, but not with threonine or methionine. The fivefold increase in soluble threonine in isoleucine-supplemented plants suggests that threonine dehydratase is a major locus for feedback regulation of isoleucine synthesis. It is concluded that regulation of threonine biosynthesis differs from that of the other amino acids of the aspartate family (isoleucine, methionine, and lysine), each of which strongly feedback regulates its own synthesis. Methionine supplementation had a negligible effect on the tissue concentration of soluble threonine, indicating that threonine is not important in balancing changes of flux into methionine by equivalent changes of flux through the step catalyzed by aspartokinase.

Publisher Summary This chapter presents the studies on homocystinuria. Methionine is converted via S-adenosylmethionine and S-adenosylhomocysteine to homocysteine. Homocysteine may be methylated to form methionine. Human brain is extremely rich in cystathionine. This high concentration is specific for the tissue and for the species. Brains of a variety of other mammals have lower concentrations of cystathionine. Cystathionine synthase is present in brain at concentrations about 20% of the comparable hepatic concentrations. Certainly the brain is one of the relatively few tissues in which major amounts of cystathionine synthesis occurs. The values for cystathionase specific activity in brain are only about 1% of the values for liver. This observation suggests a partial explanation for the high concentration of cystathionine reported in monkey and human brain.

Deficiency of cystathioninie synthetase activity results in the clinical syndrome of homocystinuria. In both parents of a patient with homocystinuria, the hepatic cystathionine synthetase activity was 40 percent of that in unrelated control patients. These findings demonstrate that the metabolic error is inherited and suggest that the parents, although clinically normal, represent the heterozygous. state. A second case of homocystinuria also is shown to be associated with cystathionine synthetase deficiency.