Methionine and glutamine transport systems in D-methionine utilising revertants of Salmonella typhimurium

Summary In Salmonella typhimurium, methionine auxotrophs such as metB can use D-methionine as a methionine source. MetP mutations prevent this growth since D-methionine can enter only via the metP high-affinity methionine transport system. D-methionine utilising revertants (Dmu⁺) were selected from metB23 metP760 (HU76) following nitrosoguanidine mutagenesis. The properties of two such revertants, HU206 and HU415, indicated that reversion was not due to backmutation of the metP760 mutation. Genetic analysis indicated that each strain possessed two mutations, designated dmu and gln, in addition to the original metB23 and metP760 mutations.The dmu mutation restores ability to grow on D-methionine, partly restores D- and L-methionine transport activity, and makes the cells particularly sensitive to inhibition by L-glutamine while growing on D but not L-methionine. The growth inhibition by L-glutamine was shown to be caused by competition by L-glutamine for D-methionine transport by the high-affinity methionine system. The gln mutation greatly reduces activity of the high-affinity glutamine transport system. The Dmu⁺ strains are also partly defective in the glutamine low-affinity transport system, possibly because the partially-restored methionine high-affinity system, or a component of this system, functions in the transport of glutamine by its low-affinity system.     

Fine structure mapping and complementation studies of the metD methionine transport system in Salmonella typhimurium.

A fine structure deletion map of the metD region of the chromosome of Salmonella typhimurium responsible for a high-affinity methionine transport system has been constructed. Complementation tests involving the introduction of metD+DNA contained in a pUC8 vector into metD strains indicated the presence of four complementation groups in the metD region. This suggested that the methionine system belongs to the osmotic shock-sensitive class of transport system, and therefore should possess a periplasmic methionine-binding protein and several membrane proteins. But a deletion mutation covering all known metD point mutations did not affect the level of a methionine binding activity in osmotic shock fluids, suggesting either that the deletion did not extend into the gene encoding the binding protein, or that the binding activity is not associated with the metD system. Possible reasons for the failure to isolate mutations in the gene for the binding protein are discussed.     

Methionine sulfoxide is transported by high-affinity methionine and glutamine transport systems in Salmonella typhimurium.

Three lines of evidence indicated that methionine sulfoxide is transported by the high-affinity methionine and glutamine transport systems in Salmonella typhimurium. First, methionine-requiring strains (metE) which have mutations affecting both of these transport systems (metP glnP) were unable to use methionine sulfoxide as a source of methionine. These strains could still grow on L-methionine because they possessed a low-affinity system (or systems) which transported L-methionine but not the sulfoxide. A methionine auxotroph with a defect only in the metP system, which was dependent upon the glnP+ system for the transport of methionine sulfoxide, was inhibited by L-glutamine because glutamine inhibited the transport of the sulfoxide by the glnP+ system. Second, a metE metP glnP strain could be transduced at either the metP or glnP genes to restore its ability to grow on methionine sulfoxide. Third, the transport of [14C]methionine sulfoxide was inhibited by methionine and by glutamine in the metP+ glnP+ strain. No transport was detected in the metP glnP double-mutant strain.     

Transport of L-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of L-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris.HCl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in buffered saline. L-Methionine is actively transported in WI38 by saturable, chemicallly specific mechanisms which are temperature, pH and, in part Na+ dependent, and are reactive with both L- and D-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent KM of 21.7 micrometer and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent KM of 547 micrometer and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na+-free media or after treatment with ouabain suggested that system 1 is Na+ independent and that system 2 is Na+ dependent. Preloading of cells with unlabeled L-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled L- or D-methionine or L-phenylalanine, but not in the presence of L-arginine. L-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.     

The Escherichia coli metD locus encodes an ABC transporter which includes Abc (MetN), YaeE (MetI), and YaeC (MetQ)

We report that the genes abc, yaeC, and yaeE comprise metD, an Escherichia coli locus encoding a DL-methionine uptake system. MetD is an ABC transporter with Abc the ATPase, YaeE the permease, and YaeC the likely substrate binding protein. Expression of these genes is regulated by L-methionine and MetJ, a common repressor of the methionine regulon. We propose to rename abc, yaeE, and yaeC as metN, metI, and metQ, respectively.     

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.     

The Uptake and Metabolism of Cysteine by Giardia lamblia Trophozoites

ABSTRACT. The cysteine, cystine, methionine and sulfate uptake and cysteine metabolism of Giardia lamblia was studied. Initial experiments indicated that bathocuproine sulphonate (20 μM) added to Keister's modified TYI-S-33 medium supported the growth of G. lamblia at low L-cysteine concentration. This allowed the use of high specific activity radiolabeled L-cysteine for further studies. The analyses of L-cysteine uptake by G. lamblia indicate the presence of at least two different transport systems. The total cysteine uptake was non saturable, with a capacity of 3.7 pmoles per 10⁶ cells per min per μM of cysteine, and probably represent passive diffusion. However, cysteine transport was partially inhibited by L-methionine, D-cysteine and DL-homocysteine. indicating that another system specific for SH-containing amino acids is also present. Cysteine uptake was markedly decreased in medium without serum. In contrast to cysteine, the uptake of L-methionine and sulfate were carried out by saiurable systems with apparent K_m, of 71 and 72 μM, respectively, but the Vmax of the uptake of sulfate was six orders of magnitude lower than the Vmax of methionine uptake. Cystine was not incorporated into trophozoites. [³⁵S]-labeled L-cysteine and L-methionine, but not [³⁵S]sulfate, were incorporated into Giardia proteins, indicating that the parasite lacks the capacity to synthesize cysteine or methionine from sulfate. Neither cystathionine γ lyase nor crystathionine γ synthase activities was detected in homogenates of Giardia lamblia , suggesting that the transsulfuration pathway is not active and there is no conversion of methionine to cysteine. Our data indicate that cysteine is essential for Giardia because the parasite: a) cannot take up cystine, and b) cannot synthesize cysteine de novo.     

The metD D-methionine transporter locus of Escherichia coli is an ABC transporter gene cluster

The metD D-methionine transporter locus of Escherichia coli was identified as the abc-yaeE-yaeC cluster (now renamed metNIQ genes). The abc open reading frame is preceded by tandem MET boxes bracketed by the -10 and -35 boxes of a promoter. The expression driven by this promoter is controlled by the MetJ repressor and the level of methionine.     

Amino acid uptake as a function of differentiation in Candida albicans: studies of a non-germinative variant

The transport of four amino acids (L-methionine, L-phenylalanine, L-lysine and L-alanine) was studied during pH-regulated dimorphism in Candida albicans and its stable, non-germinative variant. The permeases responsible for uptake responded differently to differentiation and the transport activities varied during the course of morphogenesis. An increase in uptake around the time of evagination was observed in all four amino acids in both the strains studied. The uptake rates of L-methionine and L-phenylalanine were greater in fully differentiated hyphae, while the rate of L-lysine was higher in fully differentiated buds. Uptake rates of L-alanine, however, did not show any morphotypic related variation. The possible implication of these transport activities in relation to differentiation is discussed.     

A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.     

A common transport system for methionine, L-methionine-DL-sulfoximine (MSX), and phosphinothricin (PPT) in the diazotrophic cyanobacterium Nostoc muscorum

We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues l-methionine-dl-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in ³⁵S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX- and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX- and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.     

Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system.

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.     

Methionine transport in Salmonella typhimurium: evidence for at least one low-affinity transport system.

The systems which transport methionine in Salmonella typhimurium LT2 have been studied. Fourteen mutants, isolated by three different selection procedures, had similar growth characteristics and defects in the specific transport process showing a Km of 0.3 microM for L-methionine, and therefore lack the high-affinity, metP transport system. The sites of mutation in four of the mutants were shown by P1-mediated transduction to be linked (0.3 to 1.1%) with a proline marker located at unit 7 on the S. typhimurium chromosome. The high-affinity system was subject to both repression and transinhibition by methionine, and it may also be regulated by the metJ and metK genes. There appeared to be at least two additional transport systems with relatively low affinities for methionine in the metP763 mutant strain, with apparent Km values for methionine of 24 microM and approximately 1.8 mM. The latter system, with a very low affinity for methionine, was inhibited by leucine. In addition, methionine inhibited leucine transport, suggesting that one of the low-affinity methionine transport systems may actually be a leucine transport system.     

Amino acid transport in the unfertilized and fertilized mouse egg.

The transport of methionine into unfertilized and fertilized mouse eggs appears to involve active transport mechanisms with similar Vmax, Km, substrate specificity and independence from Na+. An exchange diffusion system with a similar amino acid specificity to the uptake system has also been found in both types of egg. An estimate of 6.5 fmol has been made for the size of the total internal pool of exchangeable amino acids.     

Biotransformation of D-methionine into L-methionine in the cascade of four enzymes

D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation. In the second reaction step, L-phenylalanine dehydrogenase (L-PheDH) from Rhodococcus sp. was used to convert 2- oxo-4-methylthiobutyric acid to L-methionine, and formate dehydrogenase (FDH) from Candida boidinii was added for NADH regeneration. Enzyme kinetics of all enzymes was analyzed in detail. Mathematical models for separate reactions steps, as well as for the complete system were developed and validated in the batch reactor experiments. Complete conversion of D-methionine to L-methionine was achieved. Considering that both enzymes act on different substrates, such a system could be easily employed for the synthesis of other amino acids from D-isomer, as well as from the racemate of a certain amino acid (DL-amino acid).     

Mutations Affecting the Different Transport Systems for Isoleucine, Leucine, and Valine in Escherichia coli K-12

Uptake of isoleucine, leucine, and valine in Escherichia coli K-12 is due to several transport processes for which kinetic evidence has been reported elsewhere. A very-high-affinity transport process, a high-affinity transport process, and three different low-affinity transport processes were described. In this paper the existence of these transport processes is confirmed by the isolation and preliminary characterization of mutants altered in one or more of them. The very-high-affinity transport process is missing either in strains carrying the brnR6(am) mutation or in strains carrying the brn-8 mutation. This appears to be a pleiotropic effect since other transport systems are also missing. Mutant analysis shows that more than one transport system with high affinity is present. One of them, high-affinity 1, which needs the activity of a protein produced by the brnQ gene, transports isoleucine, leucine, and valine and is unaffected by threonine. The other, high-affinity 2, which needs the activity of a protein produced by the brnS gene, transports isoleucine, leucine, and valine; this uptake is inhibited by threonine which probably is a substrate. Another protein, produced by the brnR gene, is required for uptake through both high-affinity 1 and high-affinity 2 transport systems. The two systems therefore appear to work in parallel, brnR being a branching point. The brnQ gene is located close to phoA at 9.5 min on the chromosome of E. coli, the brnR gene is located close to lac at 9.0 min, and the brnS gene is close to pdxA at 1 min. A mutant lacking the low-affinity transport system for isoleucine was isolated from a strain in which the high-affinity system was missing because of a brnR mutation. This strain also required isoleucine for growth because of an ilvA mutation. The mutant lacking the low-affinity transport system was unable to grow on isoleucine but could grow on glycylisoleucine. This mutant had lost the low-affinity transport for isoleucine, whereas those for leucine and valine were unaffected. A pleiotropic consequence of this mutation (brn-8) was a complete absence of the very-high-affinity transport system due either to the alteration of a common gene product or to any kind of secondary interference which inhibits it. Mutants altered in isoleucine-leucine-valine transport were isolated by taking advantage of the inhibition that valine exerts on the K-12 strain of E. coli. Mutants resistant both to valine inhibition (Val(r)) and to glycylvaline inhibition are regulatory mutants. Val(r) mutants that are sensitive to glycylvaline inhibition are transport mutants. When the very-high-affinity transport process is repressed (for example by methionine) the frequency of transport mutants among Val(r) mutants is higher, and it is even higher if the high-affinity transport process is partially inhibited by leucine.     

Effects of supplemental methionine on antiserum-induced dysmorphology in rat embryos cultured in vitro

BACKGROUND: Heterologous antiserum to the visceral yolk sac (AVYS) is teratogenic, inducing a spectrum of malformations in vivo and producing similar effects in vitro. Numerous studies support the concept that AVYS-induced malformations result from embryonic nutritional deficiency, without affecting the maternal nutritional status. This has provided a useful model with which to investigate the nutritional requirements of the early embryo, as well as the role of various nutrients in the etiology of congenital defects. METHODS: In the current investigation, we examined the effects of methionine and other nutrients on AVYS-induced embryotoxicity in vitro. For these experiments, we cultured rat embryos (9.5 p.c) for 48 hr with AVYS and/or methionine at several concentration levels. RESULTS: The addition of L-methionine to AVYS-exposed cultures reduced dysmorphology and open neural tube; this effect was concentration dependent. AVYS-induced dysmorphology was completely prevented at a concentration of L-methionine corresponding to 50-fold the basal serum concentration. Utilization of D-methionine, L-leucine, or folic acid (5-methyltetrahydrofolate, MTHF) instead of L-methionine had no protective effects. CONCLUSIONS: These results suggest that, although AVYS limits the supply of all amino acids to the embryo, embryopathy largely results from a deficiency of methionine. Furthermore, although endocytosis and degradation of proteins by the VYS supplies most amino acids to the embryo, free amino acids may be compensatory when this source is reduced. These results support those of previous investigations that suggest methionine is required for normal NT closure and that methionine is a limiting nutrient for embryonic development.     

Disruption of thymidylate synthesis and glycine-serine interconversion by L-methionine and L-homocystine in Raji cells

Excessive concentrations of L-methionine inhibited the folate-dependent de novo synthesis of thymidylic acid (TMP) in Raji cells, demonstrating the usefulness of this cell line for the study of methionine-folate antagonism. The effect was also produced by L-homocystine but not by other amino acids including D-methionine and L-ethionine, suggesting that this effect is exerted by a common intermediate of methionine and homocystine metabolism. L-Methionine, L-homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) are not inhibitors of thymidylate synthase activity. On the other hand the capacity of the cells to incorporate serine 3-carbon and glycine 2-carbon into DNA is impaired by the presence of L-methionine or L-homocystine. Studies with cell-free extracts demonstrated that the glycine cleavage enzyme is inhibited by 45% by L-methionine, L-homocysteine, SAM or SAH. Serine hydroxymethylase on the other hand was slightly stimulated by these sulfur-containing compounds and this stimulation was shown to occur in the intact cell as well. These findings suggest that when levels of L-methionine metabolites are elevated, there is an increase in the use of glycine to maintain the intracellular concentration of serine, which is required for homocysteine detoxification by conversion to cystathionine. The reduction in TMP synthesis caused by excess L-methionine or L-homocystine may result from increased utilization of one-carbon units for serine synthesis.     

Methionine in pattern control of Hydra

The fresh water polyp Hydra is noted for its ability to regenerate missing body parts. Transplantation experiments indicate that the control of regeneration includes signalling over long distances. These signals appear to include diffusible morphogens, activators and inhibitors. In order to elucidate the nature of such signals, tissue of polyps was homogenized and fractionated. The fractions were tested for their ability to hinder head regeneration. The active factor within these fractions was determined to be methionine. Both the active fractions and L-methionine were found to antagonize not only head regeneration but also foot regeneration. Budding, the asexual means of reproduction, is antagonized. L-methionine acts in micromolar concentrations while the stereoisomer D-methionine does not. L-methionine may act by providing a methyl group in transmethylation processes.     

Methionine transamination—metabolic function and subcellular compartmentation

Enzymatic activities catalysing the inter-conversion of L-methionine and its oxy analogue 4-methylthio-2-oxobutyric acid (2,4-KMB) were detected in the liver, skeletal muscle and heart of the laboratory rat and of sheep. In both species the highest activity of methionine transamination was found in the liver and was located in the cytoplasm and mitochondria. We propose that physiological and nutritional role of the cytoplasmic methionine transamination is amination of 2,4 KMB and formation of L-methionine while in mitochondria the activity is responsible for disposal of excess methionine is oxidised through oxidative decarboxylation of 2,4 KMB.