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     

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.     

Bacterial ethylene synthesis from 2-oxo-4-thiobutyric acid and from methionine

The ability of selected bacterial cultures to synthesize ethylene during growth in nutrient broth supplemented with methionine or 2-oxo-4-methylthiobutyric acid (KMBA) was examined. Although most cultures transformed KMBA into ethylene, only those of Escherichia coli SPAO and Chromobacterium violaceum were able to convert exogenously added methionine to ethylene. In chemically defined media, E. coli SPAO produced the highest amounts of ethylene from methionine and KMBA. This capability was affected by the nature of the carbon source and the type and amount of nitrogen source used for growth. When glutamate was used as sole source of carbon and nitrogen for growth, the activity of the ethylenogenic enzymes was reduced to 25% of that observed with cultures grown with glucose and NH4Cl. Neither methionine nor KMBA significantly affected the ethylenogenic capacity of E. coli SPAO. Menadione and paraquat, compounds that generate superoxide radicals, stimulated ethylene synthesis by harvested cells, but not by cell-free extracts of E. coli SPAO. In addition, cells of Pseudomonas aeruginosa, which produced no ethylene in culture in the presence of exogenously added KMBA, yet possessed the necessary enzymes in an active form, were able to synthesize ethylene from KMBA when incubated with menadione or paraquat.     

Formation of ethylene by Escherichia coli.

Escherichia coli strain SPA O converts methionine to ethylene by an inducible enzyme system. L-Cysteine, L-homocysteine, methionine derivatives and the sulphur-containing analogues of L-methionine also act as precursors of ethylene. Ethylene is produced by cell suspensions only in the presence of air; cell-free preparations can produce ethylene aerobically and anaerobically, but the extent to which they do so depends on the mode of culture growth. Light stimulates ethylene production by cell suspensions and its presence is essential for production by cell-free preparations. The kinetics of ethylene biogenesis and its pH and temperature optima suggest that ethylene is a secondary metabolite.     

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 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 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     

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.     

Purification and properties of benzyl alcohol dehydrogenase from a denitrifying Thauera sp.

Toluene and related aromatic compounds are anaerobically degraded by the denitrifying bacterium Thauera sp. strain K172 via oxidation to benzoyl-CoA. The postulated initial step is methylhydroxylation of toluene to benzyl alcohol, which is either a free or enzyme-bound intermediate. Cells grown with toluene or benzyl alcohol contained benzyl alcohol dehydrogenase, which is possibly the second enzyme in the proposed pathway. The enzyme was purified from benzyl-alcohol-grown cells and characterized. It has many properties in common with benzyl alcohol dehydrogenase from Acinetobacter and Pseudomonas species. The enzyme was active as a homotetramer of 160kDa, with subunits of 40kDa. It was NAD⁺-specific, had an alkaline pH optimum, and was inhibited by thiol-blocking agents. No evidence for a bound cofactor was obtained. Various benzyl alcohol analogues served as substrates, whereas non-aromatic alcohols were not oxidized. The N-terminal amino acid sequence indicates that the enzyme belongs to the class of long-chain Zn²⁺-dependent alcohol dehydrogenases, although it appears not to contain a metal ion that can be removed by complexing agents.Dedicated to Prof. Achim Trebst     

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.     

The physiology of L-methionine catabolism to the secondary metabolite ethylene by Escherichia coli

Catabolism of L-methionine by Escherichia coli strain B SPAO led to the formation of ethylene as a secondary metabolite (ethylenogenesis). Methionine was initially deaminated by a transamination reaction to the 2-oxo acid 2-oxo-4-methylthiobutyric acid (KMBA) which was then converted to ethylene. The utilization of L-methionine as an additional nitrogen source was investigated by examining ethylene synthesis under different nitrogen supply conditions. Ethylene formation in batch culture was unaffected by the concentration of the precursor L-methionine in the medium although increasing concentrations of NH4Cl resulted in progressively less ethylene formation. Cultures grown without L-methionine did not produce ethylene but were able to synthesize ethylene when L-methionine or KMBA was provided. Addition of L-tyrosine to batch cultures reduced the yield of ethylene after 42 h by 54%. Under these conditions the maximum transient level of KMBA was reduced by 32% and occurred later compared to when L-methionine was the only amino acid supplement. Continuous cultures grown under ammonia limitation produced both ethylene and KMBA. In contrast, when glucose was limiting, neither of these metabolites were produced. Cells harvested from continuous cultures grown under glucose or ammonia limitation were able to synthesize ethylene from either L-methionine or KMBA although their capacity for ethylene synthesis (ethylenogenic capacity) was optimal under ammonia limitation (C:N ratio = 20).     

Lysine biosynthesis in Rhodotorula glutinis: properties of pipecolic acid oxidase.

Pipecolic acid oxidase from Rhodotorula glutinis, which converts pipecolic acid to alpha-aminoadipic-delta-semialdehyde, an intermediate of the biosynthetic pathway of lysine, was purified 290-fold. The enzyme from the crude extract and purified preparation exhibited a molecular weight of approximately 43,000 and was composed of a single subunit. The purified enzyme was heat labile and exhibited a pH optimum of 8.5 and an apparent Km for L-pipecolic acid of 1.67 X 10(-3) M. L-Proline acted as a competitive inhibitor for the enzyme. The enzyme was inhibited by the sulfhydryl agents p-chloromercuribenzoate and mercuric chloride. The in vitro enzyme activity required oxygen and upon oxidation of pipecolic acid, oxygen was reduced to hydrogen peroxide.     

Ethylene production from methionine

1. A new reaction is described in which ethylene is formed from the Cu(+)-catalysed breakdown of methionine in phosphate buffer at 30 degrees in air. Some of the other products of the reaction are methionine sulphone, methionine sulphoxide, homocysteic acid, homocystine, acrolein, dimethyl disulphide, methanethiol, ethyl methyl sulphide, methane and ethane. These are considered to be produced in different reaction pathways. 2. Hydrogen peroxide is an intermediate in this reaction and can support ethylene production in the model system in anaerobic atmospheres. Cuprous copper is the active form that catalyses the formation of ethylene from an oxidized intermediate. The initial reaction is probably a Strecker degradation, but the aldehyde product is further degraded to ethylene and other products. 3. Methional (CH(3).S.CH(2).CH(2).CHO) is the most effective producer of ethylene in the model system and appears to be an intermediate in the reaction. 4. The evidence, from both tracer studies and from other precursors of ethylene in the reaction, indicates that ethylene is derived from the -CH(2).CH(2)- group of methionine.     

Kinetics and effects of trace elements and electron complexes on 2-keto-4-methylthiobutyric acid-dependent biosynthesis of ethylene in soil

AIMS: 2-Keto-4-methylthiobutyric acid (KMBA) is an established intermediate in microbial biosynthesis of ethylene from methionine. This study demonstrates the kinetics and effects of trace elements and electron complexes on substrate (KMBA)-derived C2H4 biosynthesis in soil. METHODS AND RESULTS: We have previously reported KMBA-dependent C2H4 production in soil. We studied the kinetics and effects of various trace elements and electron complexes on KMBA-derived C2H4 biosynthesis in soil by gas chromatography. Kinetic analysis revealed that ethylene forming enzyme (EFE) reaction was linear (R2 = 0.9448) when velocity of reaction (V) was plotted against substrate [S] over the range from 2.5 to 10 mmol l(-1) and thus followed a first order reaction. Application of three linear transformations of the Michaelis-Menten equation indicated high affinity of EFE for the substrate because Km values ranged between 5.4 and 6.67 mmol l(-1) and Vmax of reaction was between 22.4 and 35.7 nmol kg(-1) soil 120 cm(-1). Most of the trace elements exhibited positive effects on KMBA-dependent C2H4 production in soil. Maximum stimulatory effect on C2H4 biosynthesis was observed in response to Co(II) application, while Fe(III) inhibited the biotransformation of KMBA into C2H4. Contrarily, most of the tested electron complexes inhibited KMBA-derived C2H4 biosynthesis in the soil. However, lower concentrations (1.0 mmol l(-1)) of mannitol and hydroquinone were stimulatory to C2H4 production in soil compared with controls (substrate only). Conclusions: The results revealed that both kind and concentration of trace elements and electron complexes affected the substrate-dependent production of C2H4 in soil with different degrees of efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The C2H4 in the root environment could be physiologically active even at low concentrations, so knowledge regarding various factors which regulate C2H4 biosynthesis in soil could be of significance for plant growth and development.     

Biosynthesis of auxin-induced ethylene in mung bean hypocotyls

The pathway of ethylene biosynthesis in auxin-treated mung beanhypocotyls was investigated by comparing the specific radioactivitiesof ethylene produced and S-adenosylmethionine (SAM) in the tissuefollowing the administration of 3,4-¹⁴C-methionine, and by analyzingthe methionine metabolites. When the rate of auxin-induced ethyleneproduction was low due to a low concentration of auxin, thespecific radioactivity of ethylene released was always higherthan that of SAM in the tissue. When the tissue was treatedwith auxin, the tissue produced and accumulated a methioninemetabolite which was converted into ethylene more efficientlythan methionine. The metabolite was identified as 1-aminocyclopropane-l-carboxylicacid (ACC) by means of paper and thin-layer chromatography,high voltage paper electrophoresis and co-crystallization. ACCformation was neither inhibited by low oxygen nor by the inhibitoryprotein of ethylene synthesis, but inhibited by aminoethoxyvinylglycine(AVG). ACC application to the tissue greatly reduced incorporationof 3,4-¹⁴C-methionine into ethylene. The control tissue thatwas not treated with auxin also converted ACC into ethyleneindicating that the enzyme which converts ACC into ethyleneis already present in the tissue and that auxin induced productionof the enzymatic system responsible for the conversion of methionineinto ACC. Ethylene synthesis from ACC was not inhibited by AVG,abscisic acid, cycloheximide or actinomycin D, but inhibitedby low oxygen and the inhibitory protein. (Received November 21, 1979; )     

Horseradish peroxidase-catalyzed aerobic oxidation and peroxidation of indole-3-acetic acid. I. Optical spectra.

A study of the indole-3-acetate reaction with horse-radish peroxidase, in the absence or presence of hydrogen peroxide, has been performed, employing rapid scan and conventional spectrophotometry. We present here the first clear spectral evidence, obtained on the millisecond time scale, indicating that at pH 5.0 and for high [enzyme/substrate] ratios peroxidase compound III is formed. Most, if not all, of the compound III is formed by oxygenation of the ferrous peroxidase. There is an inhibitory effect of superoxide dismutase and histidine on compound III formation which indicates the involvement of the active oxygen species superoxide and singlet oxygen. It is concluded that the oxidation of indole-3-acetate by horseradish peroxidase at pH 5.0 proceeds through compound III formation to the catalytically inactive forms P-670 and P-630. A reaction path in which the enzyme is directly reduced by indole-3-acetate might be involved as an initiation step. Rapid scan spectral data, which indicate differences in the formation and decay of enzyme intermediate compounds at pH 7.0, in comparison with those observed at pH 5.0, are also presented. At pH 7.0 compound II is a key intermediate in oxidation--peroxidation of substrate. Mechanisms of reactions consistent with the experimental data are proposed and discussed.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeni

Aims:  Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine.
Methods and Results:  Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni .
Conclusion:  This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni . Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed.
Significance and impact of the study:  This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways.     

Synthesis of 5-S-L-cysteinyl-glycine-L-dopa, a natural substrate for serum and melanocyte dipeptidase

A method for synthesis of the phaeomelanin pigment intermediate compound 5-S-L-cysteinyl-glycine-L-dopa is presented. This thioether has been suggested as a precursor to 5-S-L-cysteinyl-L-dopa, the key intermediate compound in phaeomelanin pigment formation. 5-S-Glutathione-L-dopa is first synthesized by the tyrosinase-catalyzed reaction between L-dopa and glutathione. The 5-S-glutathione-L-dopa is then converted to 5-S-L-cysteinyl-glycine-L-dopa using the enzyme gamma-glutamyl transpeptidase. The compound thus obtained was suitable as a substrate for melanoma cell and serum dipeptidase which converts the compound into 5-S-L-cysteinyl-L-dopa and glycine. The optimum pH for the dipeptidase from melanoma cells was 7.5 and the Km was 1.2 mM.     

Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum

Extracts from the unicellular green alga Selenastrum capricornutum exhibit high superoxide dismutase activity, but only traces of catalase activity. The excess hydrogen peroxide (HO) generated by the superoxide dismutase in S. capricornutum may be degraded by a unique peroxidase. This peroxidase has a high specificity for ascorbate as its electron donor. The enzyme has an optimum pH at 8, is insensitive to cyanide and is inhibited by oxine. Addition of low concentrations of copper to algal cultures stimulates the peroxidase activity threefold. This enzymatic system could be used as a sensitive bioindicator for copper in fresh water.