Methyl Carbon Of Acetate Research Articles

Quantification of carbon flow from stable isotope fractionation in rice field soils with different organic matter content

Rice fields are an important source for the greenhouse gas methane produced by acetoclastic and hydrogenotrophic methanogenesis. Fractionation of 13C/ 12C can in principle be used to quantify the relative contribution of these pathways, but our knowledge of isotopic fractionation during reduction of CO 2 and turnover of acetate in different methanogenic environments is still scarce. We therefore measured δ 13C signatures in two types of anoxic Italian rice field soils, one with high and one with low degradable organic matter (OM) content. Both soils were incubated in the presence and absence of methyl fluoride, a specific inhibitor of acetoclastic methanogenesis. Optimization of methyl fluoride concentration resulted in complete inhibition of acetoclastic methanogenesis. CH 4 was then exclusively produced by hydrogenotrophic methanogenesis, allowing determination of the isotopic signatures and fractionation factors specific for this methanogenic pathway. Acetate, which was then no longer consumed, accumulated and was used for determination of the isotopic signature of the fermentatively produced acetate (both total acetate and methyl carbon of acetate). Hence, all isotopic signatures, including fractionation factors were determined for the methanogenic soil. These data, were then used for computation of the relative contribution of the two methanogenic pathways. In the high OM soil, the contribution of acetoclastic methanogenesis to total CH 4 production increased simultaneously with decreasing acetate concentration. In the low OM soil, methanogenesis from H 2/CO 2 was clearly greater than theoretically expected. Furthermore, isotope fractionation of hydrogenotrophic methanogenesis indicated that the in situ energy status of methanogens strongly depended on the availability of organic carbon in the rice field soil system. Collectively, our data show that the study of isotopic fractionation in methanogenic environments allows a deeper insight into the ongoing processes, which may be quite different in the same ecosystem with different content of degradable OM.

Solution (sup13)C Nuclear Magnetic Resonance Spectroscopic Analysis of the Amino Acids of Methanosphaera stadtmanae: Biosynthesis and Origin of One-Carbon Units from Acetate and Carbon Dioxide

We found that general pathways for amino acid synthesis of Methanosphaera stadtmanae, a methanogen that forms CH(inf4) from H(inf2) and methanol, resembled those of methanogens that form CH(inf4) from CO(inf2) or from the methyl group of acetate. We determined the incorporation of (sup14)C-labeled CO(inf2), formate, methanol, methionine, serine, and acetate into cell macromolecules. Labeling of amino acid carbons was determined by solution nuclear magnetic resonance spectroscopy after growth with (sup13)C-labeled acetate, CO(inf2), serine, and methanol. The (alpha) and (beta) carbons of serine and alanine were formed from carboxyl and methyl carbons of acetate, respectively, and the amino acid carboxyl groups were formed from CO(inf2). This indicates that pyruvate was formed by reductive carboxylation of acetate. Labeling of the methyl carbon of methionine indicated that the major route of synthesis was from the hydroxymethyl carbon of serine that arises from the methyl carbon of acetate. Methanol was a minor source of the methyl of methionine. Unambiguous assignment was made of the sources of all carbons of histidine. Labeling of the histidine 7 position ((epsilon) carbon) was consistent with formation from the C-2 of the purine ring of ATP and the origin of the C-2 from a formyl unit derived from the hydroxymethyl carbon of serine.

Biosynthesis of 5-hydroxy-4-oxo-L-norvaline in Streptomyces akiyoshiensis

The biosynthesis of 5-hydroxy-4-oxo-L-norvaline (HON) in Streptomyces akiyoshiensis has been investigated using 13C-labelled substrates. Incorporations of 13C label from sodium [1-13C]-, [2-13C]-, and [1,2-13C2]acetate indicated that HON was formed from a four-carbon compound derived from the citric acid cycle and the methyl carbon of acetate. Feeding experiments using DL-[4-13C]- and DL-[2-13C,15N]aspartate demonstrated that aspartate served as the four-carbon precursor to HON. Both enantiomers of aspartate were metabolized by S. akiyoshiensis, but the D isomer was consumed at a slower rate. The distribution of 13C label in the intracellular L-glutamic acid isolated in these feeding experiments is consistent with the operation of the citric acid cycle in S. akiyoshiensis. A biosynthetic hypothesis that involves a condensation reaction between acetyl or malonyl CoA and the β-carboxyl group of aspartate, and subsequent oxidative decarboxylation, is proposed to account for the incorporation results. An analogous condensation step has been proposed for the biosynthesis of other natural products, including the carbapenem antibiotics. DL-[2-13C,15N]Aspartate was synthesized from [2-13C]diethylmalonate and potassium [15N]phthalimide via diethyl [2-13C,15N]phthalimidomalonate.

Metabolites produced by the Scleroderris canker fungus, Gremmeniellaabietina. Part 4. Biosynthetic studies

The biosynthetic origin of the carbon atoms of the most highly oxidized, polyketide derived, ring (carbons 1–3) of the Gremmeniellaabietina metabolites sclerodione (3), scleroderolide (4), sclerodin (5), and Scleroderris green (6) has been studied. It is shown that both C-1 and C-3 of 3 and 4 are derived from C-1 of acetate, and that C-2 of scleroderolide (4) is derived from C-2 of acetate while C-3 is derived from C-1 of acetate. As expected, C-1 and C-3 of Scleroderris green (6) originate from the carboxyl carbon of acetate, while C-2 originates from the methyl carbon of acetate. Complete 13C and 1H nuclear magnetic resonance spectra of these metabolites are reported. It has also been shown that ent-atrovenetin (ent-7a) is a metabolite of G. abietina.

Metabolism of propionate to acetate in the cockroach Periplaneta americana

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5- 13C 3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2- 13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3- 13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2- 13C]propionate was readily metabolized to acetate. The radioactivity from sodium [1- 13C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1- 13C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.

Biosynthesis of slaframine, (1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine, a parasympathomimetic alkaloid of fungal origin. 3. Origin of the pyrrolidine ring.

The phytopathogen Rhizoctonia leguminicola has previously been shown to incorporate pipecolic acid into the piperidine alkaloids 1-acetoxy-6-aminooctahydroindolizine (slaframine) and 3,4,5-trihydroxyoctahydro-1-pyrindine. In the experiments described here, resting cultures of R. leguminicola were incubated with [1-14C]- and [2-14C]malonic acid and with [1-14C]- and [2-2H]acetic acid. Both acids were incorporated into the ring systems of both alkaloids. Mass spectrometric analysis of 2H-enriched slaframine showed that the label resides in the five-membered ring and that the methyl carbon of acetate is joined to the carboxyl carbon of pipecolate. A pipecolate-dependent decarboxylation of [1-14C]malonate was demonstrated in cell-free extracts of R. leguminicola. The results account for previously unattributed carbons in the two alkaloids and suggest the formation of an eight-carbon intermediate common to both alkaloids by acylation of malonate with pipecolic acid.

Acetate metabolism in Methanosarcina barkeri.

Methanosarcina barkeri was grown by acetate fermentation in complex medium (N2 gas phase). The molar growth yield was 1.6–1.9 g cells/mol methane formed. Under these conditions 63–82% of the methane produced byMethanosarcina strains was derived from the methyl carbon of acetate, indicating that some methane was derived from other media components. Growth was not demonstrated in complex media lacking acetate or mineral acetate medium containing acetate but lacking H2/CO2, methanol, or trypticase and yeast extract. Acetate metabolism byM. barkeri strain MS was further exmined in mineral acetate medium containing H2/CO2 and/or methanol, but lacking cysteine. Under these conditions, more methane was derived from the methyl carbon of acetate than from the carboxyl carbon. Methanogenesis from the methyl group increased with increasing acetate concentration. The methyl carbon contributed up to 42% of the methane formed with H2/CO2 and up to 5% with methanol. Methanol stimulated the oxidation of the methyl group of acetate to CO2. The average rates of methane formation from acetate were 1.3 nomol/min ·ml/culture (0.04mg2 cell dry weight) in defined media (gas phase H2/CO2) and complex media (gas phase N2). Acetate contributed up to 60% of cell carbon formed under the growth conditions examined. Similar quantities of cell carbon were derived from the methyl and carboxyl carbons of acetate, suggesting incorporation of this compound as a two-carbon unit. Incorporated acetate was not preferentially localized in lipid material, as 70% of the incorporated acetate was found in the wall and protein cell fractions. Acetate catabolism was stimulated by pregrowing of cultures in media containing acetate, while acetate anabolism was not influenced. The results are discussed in terms of the differences between the mechanisms of acetate catabolism and anabolism.

A new thioglucoside, 2-methylpropylglucosinolate

Based on relative efficiencies of conversion of 14C from feeding labeled compounds to Conringia orientalis (L.) Andrz., the aglycone moiety of 2-hydroxy-2-methylpropylglucosinolate has been found to be formed from valine and the methyl carbon of acetate via leucine. Isobutyl isothiocyanate has been identified as the aglycone formed upon myrosinase hydrolysis of a new thioglucoside, 2-methylpropylglucosinolate, present in fresh plants of C. orientalis. It is suggested that 2-methylpropylglucosinolate may be an intermediate in the biosynthetic pathway between leucine and 2-hydroxy-2-methylpropylglucosinolate.

BIOSYNTHESIS OF MUSTARD OIL GLUCOSIDES: I. ADMINISTRATION OF C14-LABELLED COMPOUNDS TO HORSERADISH, NASTURTIUM, AND WATERCRESS

Biosynthetic investigations with C14-labelled compounds indicate that the aromatic isothiocyanate moieties of mustard oil glucosides obtained from garden nasturtium (Tropaeolum majus L.) and watercress (Nasturtium officinale R.Br.) are derived from phenylalanine. Similar investigations on sinigrin, the mustard oil glucoside isolated from horseradish (Armoracia lapathifolia Gilib.) demonstrate that glycine is incorporated into allyl isothiocyanate. The methyl carbon of acetate was readily incorporated into sinigrin and gluconasturtium and was found almost exclusively in the 'isothiocyanate carbon'; on the other hand the carboxyl carbon is a poor precursor of sinigrin.

BIOSYNTHESIS OF MUSTARD OIL GLUCOSIDES: I. ADMINISTRATION OF C14-LABELLED COMPOUNDS TO HORSERADISH, NASTURTIUM, AND WATERCRESS

Biosynthetic investigations with C14-labelled compounds indicate that the aromatic isothiocyanate moieties of mustard oil glucosides obtained from garden nasturtium (Tropaeolum majus L.) and watercress (Nasturtium officinale R.Br.) are derived from phenylalanine. Similar investigations on sinigrin, the mustard oil glucoside isolated from horseradish (Armoracia lapathifolia Gilib.) demonstrate that glycine is incorporated into allyl isothiocyanate. The methyl carbon of acetate was readily incorporated into sinigrin and gluconasturtium and was found almost exclusively in the 'isothiocyanate carbon'; on the other hand the carboxyl carbon is a poor precursor of sinigrin.

In vivo Studies of Methanogenesis in the Bovine Rumen: Dissimilation of Acetate

SUMMARY: The introduction of sodium acetate-2-14C into a bovine rumen resulted in the in vivo labelling of the rumen gases and volatile fatty acids. The relative isotope concentration in substrate and products indicated that a maximum of 5.6% of the methane and 11% of the carbon dioxide in rumen gas might have been derived from the methyl carbon of acetate when the substrate was added to the rumen 18 hr. after the animal had been fed a normal ration. A maximum of 3.2% of the methane and 4.2% of the carbon dioxide might have been derived from the methyl carbon of sodium acetate-2-14C when this substrate was introduced into the rumen immediately after the animal had been fed. The addition of sodium acetate-1-14C to the rumen 18 hr. after feeding indicated that 2% of the methane and 10% of the carbon dioxide was derived from the carboxyl carbon of the substrate. Most of the derived radioactivity of the volatile acids of the rumen was found in the butyric acid fraction, although smaller amounts appeared in propionic acid and the volatile fatty acids with a chain length of greater than 4 carbons.

On the Biosynthesis of Gibberellins from Carbon-14-Substrates by Fusarium moniliforme

SUMMARYConditions are described under which C14-labeled gibberellic acid was synthesized by cultures of Fusarium moniliforme (NRRL 2284) from acetate-1-C14 and acetate-2-C14. The results showed that the methyl-carbon of acetate was converted more efficiently to gibberellic acid than the carboxyl-carbon. Also, 3-methyl crotonic acid-3-C14 and 3-methyl crotonic acid-3-methyl-C14, 4-C14 were converted into small quantities of C14-labeled gibberellic acid. Other substrates which were studied included mevalonic acid-2-C14, uniformly C14-labeled sucrose, glucose, and fructose. These experiments confirmed that acetate and 3-methyl crotonic acid may be precursors in the biosynthesis of gibberellic acid by F. moniliforme.

Studies on the metabolism of acetate by acetate-requiring mutants of Neurospora crassa

1. 1. Resting cultures of an acetate-requiring mutant accumulate pyruvic acid and acetylmethylcarbinol when shaken in buffer with glucose or acetate. 2. 2. The methyl carbon of acetate is incorporated into the pyruvate and the acetylmethylcarbinol; the carboxyl carbon enters the pyruvate but not the acetylmethylcarbinol. Neurospora can incorporate CO 2 into pyruvic acid. 3. 3. Sodium fluoroacetate inhibits acetate oxidation and mycelial growth. Fluoroacetate-inhibited cultures accumulate citrate. 4. 4. The methyl carbon of acetate appears as CO 2 as readily in acetate-requiring mutants which are unable to oxidize pyruvic acid as in the wild-type. 5. 5. “Succinate” mutants will grow adaptively on much smaller quantities of acetate than required by the “acetate” mutants. 6. 6. It is considered likely that Neurospora ordinarily oxidizes acetate via a tricarboxylic acid cycle, but that a dicarboxylic acid cycle might operate under certain conditions. 7. 7. Acetate inhibits the accumulation of pyruvate from glucose by acetate-requiring mutants.