Studies of ethylene-forming system in rat liver extract.

Evidence of enzymatic formation of ethylene from methionine by rat liver extracts is presented. The ethylene production is closely associated with growth of the animal. The conversion of L-methionine to ehtylene is oxygen dependent. Substrate analogue studies show that the ethylene-forming system is structurally specific and requires in the center of the molecule alpha-CH2-CH2- with one end attached to an unencumbered sulfur atom from a thioether moiety and the other end attached to a carboxyl group. Sylfhydryl agents are found to be very effective inhibitors of the ethylene-forming system. The finding of alpha-keto-4-methylthiobutyric acid to be a more efficient precursor of ethylene production suggests the possibility that alpha-keto-4-methylthiobutyric acid may be an intermediate in the biosynthesis of ethylene from methionine in mammalian tissues.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Ethylene production from l-methionine by Cryptococcus albidus

Cryptococcus albidus IFO 0939 was selected from microorganisms producing ethylene from l-methionine in a culture medium. When methionine was excluded from the culture medium of C. albidus, there was little production of ethylene. Ethylene production in a methionine-containing culture medium occurred for a brief period at the end of the growth phase. 2-oxo-4-methylthiobutyric acid (KMBA), a deaminated product of methionine, accumulated in the culture filtrate. An ethylene-forming enzyme was partially purified from C. albidus by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system, the precursor of ethylene was found to be KMBA and essential factors were NAD(P)H, Fe³⁺, EDTA and oxygen.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Metabolism of 5-methylthioribose to methionine

During ethylene biosynthesis, the H₃CS- group of S-adenosylmethionine is released as 5′-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, [¹⁴C]MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of [ribose-U-¹⁴C]MTR with avocado extract resulted in the production of [¹⁴C]formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, l-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O₂ in the conversion of MTR to methionine is discussed.     

An evaluation of 4-s-methyl-2-keto-butyric Acid as an intermediate in the biosynthesis of ethylene

Stimulation of ethylene production by cauliflower (Brassica oleracea var. botrytis L.) tissue in buffer solution containing 4-S-methyl-2-keto-butyric acid is not due to activation of the natural in vivo system. Increased ethylene production derives from an extra-cellular ethylene-forming system, catalyzed by peroxidase and other factors, which leak from the cauliflower tissue and cause the degradation of 4-S-methyl-2-keto-butyric acid. This exogenous ethylene-forming system is similar to the ethylene-forming horseradish peroxidase system which utilizes methional or 4-S-methyl-2-keto-butyric acid as substrate. We conclude that 4-S-methyl-2-keto-butyric acid is probably not an intermediate in the biosynthetic pathway between methionine and ethylene.     

Stimulation of ethylene production in tomato tissue by propionic Acid

Propionic acid (10⁻³m) increases ethylene production by about 30 to 60% in tissue from green and half-ripe tomatoes (Lycopersicon esculentum Mill. var. Homestead) but does not increase ethylene production in tissue from ripe fruit. Stimulation is not due to the conversion of propionic acid to ethylene but appears to be secondary in nature and to operate on the endogenous ethylene-forming system. Thus conversion of methionine to ethylene in green and half-ripe tomato tissue is increased in the presence of propionic acid. Inhibitors which affect the normal endogenous ethylene-forming system similarly affect the propionic acid-stimulated system. Endogenous propionic acid may play a role in the regulation of ethylene production in tomato tissues.     

The metabolism of 5'-methylthioadenosine and 5-methylthioribose 1-phosphate in Saccharomyces cerevisiae

Cordycepin sensitive mutants of Saccharomyces cerevisiae, which are permeable to 5'-deoxy-5'-methylthioadenosine (MTA), were used to study the fate of the methylthioribose carbons of this purine nucleoside. Evidence is presented for the recycling of the methylthio group and part of the ribose portion of MTA in a biosynthetic pathway which leads to the synthesis of methionine. The main pathway involves the phosphorylytic cleavage of MTA by MTA phosphorylase yielding 5-methylthioribose 1-phosphate and adenine as products. Loss of the phosphate group of 5-methylthioribose 1-phosphate, concurrent with the rearrangement of the ribose carbons, leads to the synthesis of 2-keto-4-methylthiobutyric acid. In the final step of the sequence, 2-keto-4-methylthiobutyric acid is converted to methionine via transamination. Several compounds not directly associated with the biosynthesis of methionine were also isolated. These compounds, which may arise through the degradation of intermediates in the pathway, were: 5'-methylthioinosine, a deaminated catabolite of MTA; 5-methylthioribose, a result of the phosphorylysis of 5-methylthioribose 1-phosphate, and 3-methylthiopropionaldehyde, 3-methylthiopropionic acid and 2-hydroxy-4-methylthiobutyric acid, all arising from the catabolism of 2-keto-4-methylthiobutyric acid.     

Methylthioadenosine metabolism in malignant murine cells

An ether-extractable product formed from 5′-methylthioadenosine by extracts of malignant murine lymphocytic cells is shown to be 2-keto-4-methylthiobutyric acid. When 5′-methylthio [U-¹⁴C]adenosine was used as substrate, the product was labelled, confirming earlier reports that carbons of the keto acid are derived from carbons of the ribose. When hydroxylamine was added to the reaction mixture, the ketomethylthiobutyric acid was trapped as the oxime. When glutamine was added, the main product was methionine.     

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.     

Inhibition of Ethylene Production in Penicillium digitatum

Production of ethylene by static cultures of Penicillium digitatum, which utilize glutamate and α-ketoglutarate as ethylene precursors, was inhibited by methionine, methionine sulfoxide, methionine sulfone, and methionine sulfoximine. Rhizobitoxine did not affect ethylene production but its ethoxy and methoxy analogues were effective inhibitors of ethylene production; its saturated methoxy analogue and kainic acid stimulated ethylene production. Tracer studies showed that the inhibitors blocked the conversion of [³H]glutamate into [³H]ethylene.

In shake cultures of this fungus, which utilize methionine as the ethylene precursor, rhizobitoxine and its unsaturated analogues all inhibited, while the saturated methoxy analogue stimulated ethylene production. In both types of cultures inhibition was irreversible and was diminished by increasing concentrations of the ethylene precursor. The inhibitory activity or lack of it by rhizobitoxine and its analogues appears to be a function of their structural resemblance to glutamate and methionine as well as of their size and stereoconfiguration. These data suggest similarities between the ethylene-forming system in the fungus and in higher plants despite differences in precursors under some cultural conditions.

     

Evaluation of the role of methional, 2-keto-4-methylthiobutyric acid and peroxidase in ethylene formation by Escherichia coli.

During growth of Escherichia coli strain SPA O in the presence of methionine, an intermediate accumulates in the medium. This intermediate reacts with 2,4-dinitrophenylhydrazine, and can be degraded to ethylene either enzymically or photochemically, the latter being stimulated by the addition of a flavin. The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthiobutyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6. The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis. The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate. A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction. Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed.     

Ketomethylthiobutyric acid formation from methylthioadenosine: A diffusion assay

Typical enzyme kinetics were observed when 5′-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H₂S derived from the cysteine. The yield of H₂S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5′-methylthioadenosine + P_i → adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P → KMTB, (3) KMTB + cysteine → methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH → pyruvate + H₂S, (5) H₂S + 5,5′-dithiobis(2-nitrobenzoic acid) → 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of α-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol.     

Stimulation of ethylene production in apple tissue slices by methionine

Methionine can induce more than a 100% increase in ethylene production by apple tissue slices. The increased amount of ethylene derives from carbons 3 and 4 of methionine. Only post-climacteric fruit tissues are stimulated by methionine, and stimulation is optimum after 8 months' storage. Copper chelators such as sodium diethyl dithiocarbamate and cuprizone very markedly inhibit ethylene production by tissue slices. Carbon monoxide does not effect ethylene production by the slices. These data suggest that the mechanism for the conversion of methionine to ethylene, in apple tissues, is similar to the previously described model system for producing ethylene from methionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systems in tissues derives from methionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase.     

Ethylene production and toxigenicity of methionine and its derivatives with riboflavin in cultures of Verticillium, Fusarium and Colletotrichum species exposed to light

Ethylene was produced by Verticillium dahliae Kleb. grown in liquid Czapek's medium. The rate of ethylene production was enhanced by light but was not affected by shaking or the growth rate of the cultures. L-, D- and DL-methionine, DL-ethionine and a -keto- y -methylthiobutyric acid (KMBA) were good substrates for ethylene production. KMBA may be an intermediate in ethylene production and it appears to be degraded to ethylene either enzymatically by peroxidase or photochemically in the presence of riboflavin. Addition of riboflavin or briefly heating the cultures to 100°C enhanced ethylene production greatly, while the addition of sodium azide, potassium cyanide and catalase were very inhibitory. The SS4 (non-defoliating) pathotype of V. dahliae produced significantly more ethylene (up to 108.4 nl ethylene h₁ from 20 ml-10-day-old cultures) than did the T9 (defoliating) pathotype with all substrates tested. The results suggest that the in vitro rate of ethylene production is not related to the relative virulence of pathotypes of V. dahliae on cotton. A number of Verticillium species, Fusarium oxysporum f. sp. vasinfectum and Colletotrichum dematium var. truncatum were able to produce ethylene in liquid Czapek's medium containing 1 m M L-methionine under continuous light. Riboflavin, although highly stimulatory to ethylene production, caused a fungicidal reaction to all the fungi tested in Czapek's medium containing L-methionine under continuous light. The fungicidal effect of the riboflavin-methionine-light combination occurred at concentrations of riboflavin and methionine less than 1.33 μ M and 0.5 m M , respectively. No fungicidal activity was detected when the cultures were grown in total darkness or when either methionine or riboflavin was omitted from the culture medium.     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

Ethylene biosynthesis in fruit tissues

Tracer studies with avocado tissues indicate that methionine is converted to ethylene at stages of the climacteric rise and the climacteric peak, but not at the preclimacteric stage. The results suggest that the control of ethylene biosynthesis is at a step after methionine is synthesized. The endogenous content of methionine was found to be so low that methionine must be actively turned over for ethylene biosynthesis during the stages when the rate of ethylene production is high. Oxygen was found to be essential for this conversion, indicating that at least one of the steps in conversion of methionine to ethylene is oxygen-dependent. The ability of methionine and its keto analogue (α-keto-γ-methylthiobutyric acid) to serve as ethylene precursors by apple tissues was compared. Chemical and kinetic evidence support the view that methionine is a closer precursor of ethylene than its keto analogue.     

An NADH: Fe(III) EDTA oxidoreductase from Cryptococcus albidus: an enzyme involved in ethylene production in vivo?

An ethylene-forming enzyme which forms ethylene from 2-oxo-4-methylthiobutyric acid (KMBA) was purified to an electrophoretically homogeneous state from a cell-free extract of Cryptococcus albidus IFP 0939. The presence of KMBA, NADH, Fe(III) chelated to EDTA and oxygen were essential for the formation of ethylene. When ferric ions, as Fe(III)EDTA, in the reaction mixture were replaced by Fe(II)EDTA under aerobic conditions, the non-enzymatic formation of ethylene was observed. Under anaerobic conditions in the presence of Fe(III)EDTA and NADH, the enzyme reduced 2 mol of Fe(III) with 1 mol of NADH to give 2 mol of Fe(II) and 1 mol NAD+, indicating that the ethylene-forming enzyme is an NADH-Fe(III)EDTA oxidoreductase. The role of NADH:Fe(III)EDTA oxidoreductase activity in the formation in vivo ethylene from KMBA is discussed.     

Ethylene release from green spruce needles involves a combination of ACC-dependent and independent pathways

Aminoethoxyvinylglycine (AVG) and cobalt ions strongly inhibit the conversion of added methionine or aminocyclopropane-1-carboxylic acid (ACC) into ethylene by green-coloured, non-stressed Norway spruce (Picea abies L.) needles but only 30%–40% of basal ethylene formation is affected by such inhibitors. In addition, free radical-mediated ACC-independent ethylene formation (AIEF) of the type released by brown-coloured spruce needles also occurs in extracts from healthy green-coloured needles. Treatment with CdCl₂ (10 mM), Na₂S₂O₅ (5 mM) or FeSO₄ (10 mM) induces 3–7 fold increases in the rates of ethylene evolution from green-coloured needles. However, only Cd²⁺-induced ethylene formation is inhibited by AVG while ethylene induced by S₂O₅ ^2- or Fe²⁺ is insensitive to added AVG although increased levels of ACC have also been detected in these treatments. Nevertheless, ethylene-forming decomposition of the precursors of AIEF is accelerated by S₂O₅ ^- or Fe²⁺ which indicates that the ethylene released from green-coloured spruce needles is formed by a combination of both the ACC-dependent and AIEF pathways.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - AIEF ACC-independent ethylene formation - EFE ethylene-forming enzyme - MACC N-malonyl(amino)cyclopropane-1-carboxylic acid - DTBN di-tert-butylnitroxide - MNP 2-methyl-2-nitrosopropane - SAM S-adenosylmethionine - TEMPO 2,2,6,6-tetramethyl-1-piperidine-N-oxyl