Formation of ethionine from homocysteine and of S-methylmethionine from methionine in apple tissue

Conversion of l-homocysteine into ethionine and of methionine into S-methylcysteine in apple tissues is demonstrated.     

Methionine metabolism in apple tissue: implication of s-adenosylmethionine as an intermediate in the conversion of methionine to ethylene

If S-adenosylmethionine (SAM) is the direct precursor of ethylene as previously proposed, it is expected that 5′-S-methyl-5′-thioadenosine (MTA) would be the fragment nucleoside. When [Me-¹⁴C] or [³⁵S]methionine was fed to climacteric apple (Malus sylvestris Mill) tissue, radioactive 5-S-methyl-5-thioribose (MTR) was identified as the predominant product and MTA as a minor one. When the conversion of methionine into ethylene was inhibited by _l-2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid, the conversion of [³⁵S] or [Me¹⁴C]methionine into MTR was similarly inhibited. Furthermore, the formation of MTA and MTR from [³⁵S]methionine was observed only in climacteric tissue which produced ethylene and actively converted methionine to ethylene but not in preclimacteric tissue which did not produce ethylene or convert methionine to ethylene. These observations suggest that the conversion of methionine into MTA and MTR is closely related to ethylene biosynthesis and provide indirect evidence that SAM may be an intermediate in the conversion of methionine to ethylene.     

Methionine Biosynthesis is Essential for Infection in the Rice Blast Fungus Magnaporthe oryzae

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.     

Comparison of the effects of buthioninesulfoximine and phorone on the metabolism of sulfur-containing amino acids in rat liver

Alterations in hepatic transsulfuration reactions were determined in rats treated with a glutathione-depleting agent. A dose of l-buthionine-(S, R)-sulfoximine decreased hepatic methionine, cysteine, S-adenosylmethionine, and glutathione levels rapidly. Methionine adenosyltransferase and γ-glutamylcysteine lygase activities were decreased transiently, but significantly. The activity of cysteine dioxygenase was increased, resulting in an elevation of hypotaurine and taurine concentrations. Administration of phorone reduced hepatic glutathione and cysteine similarly, but S-adenosylmethionine concentrations were elevated for as long as 72 h. Hepatic methionine adenosyltransferase, cystathionine β-synthase, cystathionine γ-lyase, and γ-glutamylcysteine lygase activities were all increased but cysteine dioxygenase activity and taurine generation were markedly depressed. The results show that a decrease in hepatic GSH induces profound changes in sulfur amino acid metabolomics, which would subsequently influence various cellular processes. It is suggested that the change in hepatic levels of sulfur-containing substances and its physiological significance should be considered when a glutathione-depleting agent is utilized in biological experiments.     

Two transsulfurylation pathways in Klebsiella pneumoniae

In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-gamma-synthase and cystathionine-beta-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-beta-synthase and cystathionine-gamma-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions.     

Methionine synthesis from 3-methylthioribose in apple tissue

The primary fate of 5-methylthioribose in apple tissue is the formation of methionine. Using dual labeled 5-methylthioribose, it was shown that both the CH₃S- group and the ribose portion of 5-methylthioribose were equally incorporated into methionine. Thus, the pathway involves modification of the ribose portion of 5-methylthioribose into the 2-aminobutyrate portion of methionine. This pathway functions to recycle methionine for continued synthesis of ethylene in fruit tissues. The methionine cycle in relation to ethylene biosynthesis is presented.     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

Regulation of Auxin-induced Ethylene Production in Mung Bean Hypocotyls: Role of 1-Aminocyclopropane-1-Carboxylic Acid

Ethylene production in mung bean hypocotyls was greatly increased by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), which was utilized as the ethylene precursor. Unlike auxin-stimulated ethylene production, ACC-dependent ethylene production was not inhibited by aminoethoxyvinylglycine, which is known to inhibit the conversion of S-adenosylmethionine to ACC. While the conversion of methionine to ethylene requires induction by auxin, the conversion of methionine to S-adenosylmethionine and the conversion of ACC to ethylene do not. It is proposed that the conversion of S-adenosylmethionine to ACC is the rate-limiting step in the biosynthesis of ethylene, and that auxin stimulates ethylene production by inducing the synthesis of the enzyme involved in this reaction.     

A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species

The ribose moiety of 5′-methylthioadenosine (MTA) is metabolized to form the four-carbon unit (2-aminobutyrate) of methionine in tomato tissue (Lycopersicon esculentum Mill., cv. Pik Red). When [U-¹⁴C-adenosine] MTA was administered to tomato tissue slices, label was recovered in 5-methylthioribose (MTR), methionine, 1-aminocyclopropane-1-carboxylic acid (ACC), C₂H₄ and other unidentified compounds. However, when [U-¹⁴C-ribose]MTR was administered, radioactivities were recovered in methionine, ACC and C₂H₄, but not MTA. This suggests that C₂H₄ formed in tomato pericarp tissue may be derived from the ribose portion of MTA via MTR, methionine and ACC. The conversion of MTR to methionine is not inhibited by aminoethoxyvinylglycine (AVG), but is O₂ dependent. These data present a new salvage pathway for methionine biosynthesis which may be important in relation to polyamine and ethylene biosynthesis in tomato tissue.     

Thiosulfate Transfer Mediated by DsrE/TusA Homologs from Acidothermophilic Sulfur-oxidizing Archaeon Metallosphaera cuprina

Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the phototrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S⁰)- and tetrathionate (S₄O₆²⁻)-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys¹⁸-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys¹⁸) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C⁹³S and DsrE3A-C¹⁰¹S retained the ability to transfer the thiosulfonate group to TusA. TusA-C¹⁸S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism.     

In vivo regulation of de novo methionine biosynthesis in a higher plant (lemna)

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 ³⁵S from ³⁵SO₄²⁻ that accumulates in cystathionine and its products, rather than by the corresponding measurements with ¹⁴C. 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.     

Alterations in the metabolomics of sulfur-containing substances in rat kidney by betaine

Earlier studies have shown that betaine administration may modulate the metabolism of sulfur amino acids in the liver. In this study, we determined the changes in the metabolomics of sulfur-containing substances induced by betaine in the kidney, the other major organ actively involved in the transsulfuration reactions. Male rats received betaine (1 %) in drinking water for 2 weeks before killing. Betaine intake did not affect betaine–homocysteine methyltransferase activity or its protein expression in the renal tissue. Expression of methionine synthase was also unchanged. However, methionine levels were increased significantly both in plasma and kidney. Renal methionine adenosyltransferase activity and S-adenosylmethionine concentrations were increased, but there were no changes in S-adenosylhomocysteine, homocysteine, cysteine levels or cystathionine β-synthase expression. γ-Glutamylcysteine synthetase expression or glutathione levels were not altered, but cysteine dioxygenase and taurine levels were decreased significantly. In contrast, betaine administration induced cysteine sulfinate decarboxylase and its metabolic product, hypotaurine. These results indicate that the metabolomics of sulfur-containing substances in the kidney is altered extensively by betaine, although the renal capacity for methionine synthesis is unresponsive to this substance unlike that of the liver. It is suggested that the increased methionine availability due to an enhancement of its uptake from plasma may account for the alterations in the metabolomics of sulfur-containing substances in the kidney. Further studies need to be conducted to clarify the physiological/pharmacological significance of these findings.     

Structural and molecular genetic insight into a widespread sulfur oxidation pathway

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a ΔdsrE mutant experiment that the cytoplasmic α₂β₂γ₂-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Isolation and identification of l-cystathionine and l-selenocystathionine from the foliage of Astragalus pectinatus

l-Cystathionine and l-selenocystathionine have been isolated from the foliage of Astragalus pectinatus. In addition to these two amino acids, some S-methylcysteine and trace amounts of Se-methyl-selenocysteine were also detected in the foliage extracts. The seeds of A pectinatus were found to contain significant amounts of all four of these amino acids plus the γ-glutamyl peptides of S-methylcysteine and Se-methylselenocysteine.     

Homocysteine Biosynthesis in Green Plants: Physiological Importance of the Transsulfuration Pathway in Lemna paucicostata

To permit an assessment of the relative contributions of the transsulfuration and the direct sulfhydration pathways for homocysteine biosynthesis, the time course of incorporation of ³⁵S from ³⁵SO₄²⁻ into various sulfur-containing compounds in Lemna paucicostata has been determined. Plants were grown with either low (4.5 micromolar) or ample (1,000 micromolar) sulfate in the medium. At the shortest labeling times, ³⁵S-cystathionine was the predominant ³⁵S-containing organic sulfur compound. The flux of sulfur into cystathionine was sufficient to sustain the known rate of methionine biosynthesis. It was calculated that transsulfuration accounted for at least 90 and 85% of the total homocysteine synthesis in low and ample sulfate-grown plants, respectively (and may have accounted for 100%). No marked rise in the ³⁵S-soluble cysteine:³⁵S-homocysteine ratio was observed even at the shortest labeling times, but it is argued that this may be due to (a) the observed compartmentation of soluble cysteine, and (b) the impracticality of using labeling times shorter than 17 seconds. Additional evidence supporting the importance of transsulfuration in Lemna is briefly described.     

Methionine metabolism and ethylene biosynthesis in senescing petals of Tradescantia

The endogenous content of methionine in isolated petals of Tradescantia was found to increase during petal senescence while the levels of S-methylmethionine and protein were found to decline. The increase in free methionine was, at least in part, the result of protein degradation. Methionine and homocysteine were shown to be intermediates in ethylene biosynthesis while S-methylmethionine was not involved. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to all floral tissues resulted in large stimulations of ethylene production. ACC was shown to be an endogenous amino acid the internal levels of which correlated positively with the rate of ethylene production. Application of l-methionine-[U-¹⁴C] led to a rapid appearance of radioactivity in both ethylene and ACC. The specific radioactivity of C-2 and C-3 of ACC and that of ethylene were found to be nearly identical which indicated that ACC was the immediate precursor of ethylene in senescing petals of Tradescantia.     

The origin of the sulfur atom in thiamine

The mode of biosynthesis of the thiazole moiety of thiamine, 4-methyl-5β-hydroxyethyl thiazole (MHET) was studied using Salmonella typhimurium as test organism. It was shown by isotope incorporation experiments, that the sulfur atom, but not carbon-3, of cysteine is incorporated into MHET, indicating a separation of the sulfur atom of cysteine from the carbon chain during incorporation. Isotope competition experiments revealed that the incorporation of [³⁵S]cysteine is not significantly diluted by the presence of methionine, homocysteine, and glutathione. No incorporation of label from [¹⁴C]glutamate and [¹⁴C]formate was observed, leaving the origin of the five-carbon unit still in doubt.     

Inhibition of ethylene production by rhizobitoxine

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorghum was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, α-keto-γ-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.     

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with ³⁵S in either the SH— or SO₃ — sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO₃-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO₃-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO₃-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO₃-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.