An allosteric mechanism for switching between parallel tracks in mammalian sulfur metabolism

Methionine (Met) is an essential amino acid that is needed for the synthesis of S-adenosylmethionine (AdoMet), the major biological methylating agent. Methionine used for AdoMet synthesis can be replenished via remethylation of homocysteine. Alternatively, homocysteine can be converted to cysteine via the transsulfuration pathway. Aberrations in methionine metabolism are associated with a number of complex diseases, including cancer, anemia, and neurodegenerative diseases. The concentration of methionine in blood and in organs is tightly regulated. Liver plays a key role in buffering blood methionine levels, and an interesting feature of its metabolism is that parallel tracks exist for the synthesis and utilization of AdoMet. To elucidate the molecular mechanism that controls metabolic fluxes in liver methionine metabolism, we have studied the dependencies of AdoMet concentration and methionine consumption rate on methionine concentration in native murine hepatocytes at physiologically relevant concentrations (40–400 µM). We find that both [AdoMet] and methionine consumption rates do not change gradually with an increase in [Met] but rise sharply (~10-fold) in the narrow Met interval from 50 to 100 µM. Analysis of our experimental data using a mathematical model reveals that the sharp increase in [AdoMet] and the methionine consumption rate observed within the trigger zone are associated with metabolic switching from methionine conservation to disposal, regulated allosterically by switching between parallel pathways. This regulatory switch is triggered by [Met] and provides a mechanism for stabilization of methionine levels in blood over wide variations in dietary methionine intake.     

Analysis of pathological defects in methionine metabolism using a simple mathematical model

Derangements in methionine metabolism are a hallmark of cancers and homocystinuria, an inborn error of metabolism. In this study, the metabolic consequences of the pathological changes associated with the key pathway enzymes, methionine adenosyl transferase (MAT), glycine N-methyl transferase (GNMT) and cystathionine beta-synthase (CBS) as well as an activation of polyamine metabolism, were analyzed using a simple mathematical model describing methionine metabolism in liver. The model predicts that the mere loss of allosteric regulation of CBS by adenosylmethionine (AdoMet) leads to an increase in homocysteine concentration. This is consistent with the experimental data on the corresponding genetic defects, which specifically impair allosteric activation but not basal enzyme activity. Application of the characteristics of transformed hepatocytes to our model, i.e., substitution of the MAT I/III isozyme by MAT II, loss of GNMT activity and activation of polyamine biosynthesis, leads to the prediction of a significantly different dependence of methionine metabolism on methionine concentrations. The theoretical predictions were found to be in good agreement with experimental data obtained with the human hepatoma cell line, HepG2.     

Novel Escherichia coli K-12 mutants impaired in S-adenosylmethionine synthesis.

S-Adenosylmethionine (AdoMet) plays a myriad of roles in cellular metabolism. One of the many roles of AdoMet in Escherichia coli and Salmonella typhimurium is as a corepressor of genes encoding enzymes of methionine biosynthesis. To investigate the metabolic effects of large reductions in intracellular AdoMet concentrations in growing cells, we constructed and examined mutants of E. coli which are conditionally defective in AdoMet synthesis. Temperature-sensitive mutants in metK, the structural gene for the S-adenosylmethionine synthetase (AdoMet synthetase) expressed in minimal medium, were constructed by in vitro mutagenesis of a plasmid-borne copy of metK. By homologous recombination, the chromosomal copy was replaced with the mutated metK gene. Both heat- and cold-sensitive mutants were examined. At the nonpermissive temperature, two such mutants had 200-fold-reduced intracellular AdoMet levels and required either methionine or vitamin B12 for growth. In the presence of methionine or vitamin B12, the mutants grew at normal rates even though the AdoMet levels remained 0.5% of wild type. A third mutant when placed at nonpermissive temperature had less than 0.2% of the normal AdoMet level and did not grow on minimal medium even in the presence of methionine or vitamin B12. All of these mutants grew normally on yeast-extract-based medium in which an alternate form of S-adenosylmethionine synthetase was expressed.     

The logic of the hepatic methionine metabolic cycle

This review describes our current understanding of the “traffic lights” that regulate sulfur flow through the methionine bionetwork in liver, which supplies two major homeostatic systems governing cellular methylation and antioxidant potential. Theoretical concepts derived from mathematical modeling of this metabolic nexus provide insights into the properties of this system, some of which seem to be paradoxical at first glance. Cellular needs supported by this network are met by use of parallel metabolic tracks that are differentially controlled by intermediates in the pathway. A major task, i.e. providing cellular methylases with the methylating substrate, S-adenosylmethionine, is met by flux through the methionine adenosyltransferase I isoform. On the other hand, a second important function, i.e., stabilization of the blood methionine concentration in the face of high dietary intake of this amino acid, is achieved by switching to an alternative isoform, methionine adenosyltransferase III, and to glycine N-methyl transferase, which together bypass the first two reactions in the methionine cycle. This regulatory strategy leads to two metabolic modes that differ in metabolite concentrations and metabolic rates almost by an order of magnitude. Switching between these modes occurs in a narrow trigger zone of methionine concentration. Complementary experimental and theoretical analyses of hepatic methionine metabolism have been richly informative and have the potential to illuminate its response to oxidative challenge, to methionine restriction and lifespan extension studies and to diseases resulting from deficiencies at specific loci in this pathway.     

Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5'-deoxy-5'-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.     

The role of methionine recycling for ethylene synthesis in Arabidopsis

The methionine (Met) cycle contributes to sulfur metabolism through the conversion of methylthioadenosine (MTA) to Met at the expense of ATP. MTA is released as a by-product of ethylene synthesis from S-adenosylmethionine (AdoMet). Disruption of the Met cycle in the Arabidopsis mtk mutant resulted in an imbalance of AdoMet homeostasis at sulfur-limiting conditions, irrespective of the sulfur source supplied to the plants. At a low concentration of 100 mum sulfate, the mtk mutant had reduced AdoMet levels and growth was retarded as compared with wild type. An elevated production of ethylene was measured in seedlings of the ethylene-overproducing eto3 mutant. When Met cycle knockout and ethylene overproduction were combined in the mtk/eto3 double mutant, a reduced capacity for ethylene synthesis was observed in seedlings. Even though mature eto3 plants did not produce elevated ethylene levels, and AdoMet homeostasis in eto3 plants did not differ from that in wild type, shoot growth was severely retarded. The mtk/eto3 double mutant displayed a metabolic plant phenotype that was similar to mtk with reduced AdoMet levels at sulfur-limiting conditions. We conclude from our data that the Met cycle contributes to the maintenance of AdoMet homeostasis, especially when de novo AdoMet synthesis is limited. Our data further showed that the Met cycle is required to sustain high rates of ethylene synthesis. Expression of the Met cycle genes AtMTN1, AtMTN2, AtMTK, AtARD1, AtARD2, AtARD3 and AtARD4 was not regulated by ethylene. This result is in contrast to that found in rice where OsARD1 and OsMTK are induced in response to ethylene. We hypothesize that the regulation of the Met cycle by ethylene may be restricted to plants that naturally produce high quantities of ethylene for a prolonged period of time.     

Multistationary and Oscillatory Modes of Free Radicals Generation by the Mitochondrial Respiratory Chain Revealed by a Bifurcation Analysis

The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism.     

Hysteretic behavior of methionine adenosyltransferase III. Methionine switches between two conformations of the enzyme with different specific activity

Methionine adenosyltransferase III (MATIII) catalyzes S-adenosylmethionine (AdoMet) synthesis and, as part of its reaction mechanism, it also hydrolyzes tripolyphosphate. Tripolyphosphatase activity was linear over time and had a slightly sigmoidal behavior with an affinity in the low micromolar range. On the contrary, AdoMet synthetase activity showed a lag phase that was independent of protein concentration but decreased at increasing substrate concentrations. Tripolyphosphatase activity, which appeared to be slower than AdoMet synthesis, was stimulated by preincubation with ATP and methionine so that it matched AdoMet synthetase activity. This stimulation process, which is probably the origin of the lag phase, represents the slow transition between two conformations of the enzyme that could be distinguished by their different tripolyphosphatase activity and sensitivity to S-nitrosylation. Tripolyphosphatase activity appeared to be the rate-determining reaction in AdoMet synthesis and the one inhibited by S-nitrosylation. The methionine concentration necessary to obtain half-maximal stimulation was in the range of physiological methionine fluctuations. Moreover, stimulation of MAT activity by methionine was demonstrated in vivo. We propose that the hysteretic behavior of MATIII, in which methionine induces the transition to a higher specific activity conformation, can be considered as an adaptation to the specific functional requirements of the liver.     

Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase

KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) is a member of a restriction-modification (R-M) system in Klebsiella pneumoniae and recognizes the sequence 5'-GGTACC-3'. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine (AdoMet) to the N(6) position of adenine residue. KpnI MTase occurs as a dimer in solution as shown by gel filtration and chemical cross-linking analysis. The nonlinear dependence of methylation activity on enzyme concentration indicates that the functionally active form of the enzyme is also a dimer. Product inhibition studies with KpnI MTase showed that S-adenosyl-l-homocysteine is a competitive inhibitor with respect to AdoMet and noncompetitive inhibitor with respect to DNA. The methylated DNA showed noncompetitive inhibition with respect to both DNA and AdoMet. A reduction in the rate of methylation was observed at high concentrations of duplex DNA. The kinetic analysis where AdoMet binds first followed by DNA, supports an ordered bi bi mechanism. After methyl transfer, methylated DNA dissociates followed by S-adenosyl-l-homocysteine. Isotope-partitioning analysis showed that KpnI MTase-AdoMet complex is catalytically active.     

Effect of nitrous oxide and methionine on hepatic S-adenosylmethionine levels and in vivo histidine oxidation in rats.

Nitrous oxide (N2O) decreased in vivo oxidation of histidine in rats fed a basal diet marginally deficient in methionine, although hepatic levels of S-adenosylmethionine (AdoMet) were not significantly altered. Excess dietary methionine increased hepatic levels of AdoMet and increased histidine oxidation. However, it did not protect histidine oxidation when the rats were treated with N2O. Parenteral administration of methionine greatly increased hepatic levels of AdoMet and increased histidine oxidation in normal and N2O treated rats. This indicates that when hepatic levels of AdoMet are greatly elevated by administration of methionine, N2O does not affect in vivo histidine oxidation.     

Methyl group metabolism in sheep

1. Sheep have a very low intake of methyl nutrients in the post-ruminant state, due to the almost complete degradation of dietary choline by rumen microorganisms, the lack of dietary creatine and the relatively low content of methionine in microbial proteins. 2. Methylneogenesis provides a major source of labile methyl groups in post-ruminant sheep and impairment of the methylneogenesis leads to a marked reduction of the labile methyl pool. 3. S-Adenosylmethionine (AdoMet) metabolism via transmethylation is most active in sheep liver and pancreas and is regulated by the availability of methionine and intracellular ratios of AdoMet to S-adenosylhomocysteine (AdoHcy). 4. Adaptive mechanisms which arise as a consequence of the poor methyl nutrition in post-ruminant sheep are a marked reduction of labile methyl catabolism and an increase in the capacity of methylneogenesis.     

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N(1) of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.     

Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit.

Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.     

Quantitative evaluation and regulation of S-adenosylmethionine-dependent transmethylation in sheep tissues

The overall rates of S-adenosylmethionine (AdoMet)-dependent transmethylation were estimated in various tissues from the initial rate of S-adenosylhomocysteine (AdoHcy) plus AdoMet accumulation after blocking hydrolysis of AdoHcy. The rates were found to differ widely among the tissues of sheep and the highest rate was in the pancreas, being 600 times higher than that in the muscle. Sheep liver possessed approximately 75% of total-body capacity for transmethylation although the transmethylation rate was approximately half that in rat liver. The minimum estimate of daily requirement of AdoMet for transmethylation for adult sheep was approximately 18 mmol, far in excess of methionine intake. Methionine loading elevated AdoMet levels only in the tissues with a high or moderate rate of transmethylation. The kinetic properties of major methyltransferases in sheep liver along with tissue distribution of AdoMet and AdoHcy suggest that transmethylation rate is subject to physiological regulation by tissue levels of AdoMet and AdoHcy.     

Homocysteine

Homocysteine does not occur in the diet but it is an essential intermediate in normal mammalian metabolism of methionine. Each compound, methionine or homocysteine, is the precursor of the other. Similarly, the synthesis of one is the mechanism for the detoxification of the other. The ubiquitous methionine cycle is the metabolic basis for this relationship. In some tissues the transsulfuration pathway diverts homocysteine from the cycle and provides a means for the synthesis of cysteine and its derivatives. Methionine, (or homocysteine) metabolism is regulated by the disposition of homocysteine between these competing sequences. Both pathways require vitamin-derived cofactors, pyridoxine for transsulfuration and both folate and cobalamin in the methionine cycle. The clinical consequences of disruption of these pathways was apparent first in rare inborn errors of metabolism that cause homocystinuria, but recent studies focus on "hyperhomocysteinemia"--a lesser metabolic impairment that may result from genetic variations, acquired pathology, toxicity and nutritional inadequacy. Hyperhomocysteinemia is an independent risk factor for thrombovascular diseases however it is not clear whether the minimally increased concentration of the amino acid is the causative agent or merely a marker for the pathology. Until we resolve that question we cannot predict the potential efficacy of therapies based on folate administration with or without additional cobalamin and pyridoxine.     

Reverse methionine biosynthesis from S-adenosylmethionine in eukaryotic cells

The intracellular ratio between methionine and its activated form S-adenosylmethionine (AdoMet) is of crucial importance for the one-carbon metabolism. AdoMet recycling into methionine was believed to be largely achieved through the methyl and the thiomethyladenosine cycles. We show here that in yeast, AdoMet recycling actually occurs mainly through the direct AdoMet-dependent remethylation of homocysteine. Compelling evidences supporting this result were obtained owing to the identification and functional characterization of two new genes, SAM4 and MHT1, that encode the yeast AdoMet-homocysteine methyltransferase and S-methylmethionine-homocysteine methyltransferase, respectively. Homologs of the Sam4 and Mht1 proteins exist in other eucaryotes, indicating that such enzymes would be universal and not restricted to the bacterial or fungal kingdoms. New pathways for AdoMet or S-methylmethionine-dependent methionine synthesis are presented.     

Metabolism of excess methionine in the liver of intact rat: an in vivo 2H NMR study

L-Methionine is the most toxic amino acid if supplied in excess, and the metabolic basis for this toxicity has been extensively studied, with varying conclusions. It is demonstrated here that in vivo 2H NMR spectroscopy provides a useful approach to the study of the hepatic metabolism of methionine in the anesthetized rat. Resonances corresponding to administered L-[methyl-2H3]methionine, and to the transmethylation product sarcosine, are observed during the first 10-min period after an intravenous injection of the labeled methionine, and the time dependence has been followed for a period of 5 h. A third resonance, assigned to the N-trimethyl groups of carnitine, phosphorylcholine, and other metabolites, becomes observable several hours after administration of the deuteriated methionine. In addition, there is a small increase in the intensity of the HDO resonance over the period of the study, which is interpreted to reflect the ultimate oxidation of the labeled sarcosine methyl group via mitochondrial sarcosine dehydrogenase. Additional small 2H resonances assigned to N1-methylhistidine and creatine could be observed in perchloric acid extracts of the livers of rats treated with the deuteriated methionine. Inhibition of the flux through the transmethylation pathway is observed in the rat pretreated with the S-ethyl analogue of methionine, ethionine. These data provide strong support for the importance of glycine transmethylation in the catabolism of excess methionine.