Cyclohexane Carboxylate and Benzoate Formation from Crotonate in Syntrophus aciditrophicus

The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 ± 0.24 mol of acetate and 0.16 ± 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U-¹³C]crotonate was metabolized to [1,2-¹³C]acetate and [1,2,3,4,5,7-¹³C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [¹³C]sodium bicarbonate formed [6-¹³C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U-¹³C]crotonate, the m/z-15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [¹³C]sodium bicarbonate and unlabeled crotonate, the m/z-15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2-¹³C]acetate and unlabeled crotonate, the m/z-15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO₂ addition, rather than the use of the intact four-carbon skeleton of crotonate.     

Characterization of propionate CoA-transferase from Ralstonia eutropha H16

In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct _Re ) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct _Re was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct _Re . In addition to already known CoA acceptors for Pct _Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct _Re . Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg^?1 min^?1). K _m values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct _Re in biotechnological polyester production.     

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of ¹³C-atoms from 1-[¹³C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.     

Metabolism of Benzoate, Cyclohex-1-ene Carboxylate, and Cyclohexane Carboxylate by “Syntrophus aciditrophicus” Strain SB in Syntrophic Association with H2-Using Microorganisms

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by “Syntrophus aciditrophicus” in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. ¹³C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-¹³C₆]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 μM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of “S. aciditrophicus” and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of “S. aciditrophicus” grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of “S. aciditrophicus”-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of “S. aciditrophicus”. These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.     

Diel Variations in Carbon Metabolism by Green Nonsulfur-Like Bacteria in Alkaline Siliceous Hot Spring Microbial Mats from Yellowstone National Park

Green nonsulfur-like bacteria (GNSLB) in hot spring microbial mats are thought to be mainly photoheterotrophic, using cyanobacterial metabolites as carbon sources. However, the stable carbon isotopic composition of typical Chloroflexus and Roseiflexus lipids suggests photoautotrophic metabolism of GNSLB. One possible explanation for this apparent discrepancy might be that GNSLB fix inorganic carbon only during certain times of the day. In order to study temporal variability in carbon metabolism by GNSLB, labeling experiments with [¹³C]bicarbonate, [¹⁴C]bicarbonate, and [¹³C]acetate were performed during different times of the day. [¹⁴C]bicarbonate labeling indicated that during the morning, incorporation of label was light dependent and that both cyanobacteria and GNSLB were involved in bicarbonate uptake. ¹³C-labeling experiments indicated that during the morning, GNSLB incorporated labeled bicarbonate at least to the same degree as cyanobacteria. The incorporation of [¹³C]bicarbonate into specific lipids could be stimulated by the addition of sulfide or hydrogen, which both were present in the morning photic zone. The results suggest that GNSLB have the potential for photoautotrophic metabolism during low-light periods. In high-light periods, inorganic carbon was incorporated primarily into Cyanobacteria-specific lipids. The results of a pulse-labeling experiment were consistent with overnight transfer of label to GNSLB, which could be interrupted by the addition of unlabeled acetate and glycolate. In addition, we observed direct incorporation of [¹³C]acetate into GNSLB lipids in the morning. This suggests that GNSLB also have a potential for photoheterotrophy in situ.     

Cyclohexane carboxylate and benzoate formation from crotonate in Syntrophus aciditrophicus

The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 +/- 0.24 mol of acetate and 0.16 +/- 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U-13C]crotonate was metabolized to [1,2-(13)C]acetate and [1,2,3,4,5,7-(13)C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [13C]sodium bicarbonate formed [6-(13)C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U-(13)C]crotonate, the m/z-15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [13C]sodium bicarbonate and unlabeled crotonate, the m/z-15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2-(13)C]acetate and unlabeled crotonate, the m/z-15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO2 addition, rather than the use of the intact four-carbon skeleton of crotonate.     

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution ¹³C NMR. The oxidation of [1,2,3-¹³C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS^?) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The ¹³C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure(TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the ¹³C spectra of cells presented with [3-¹³C] propionate or [2-¹³C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.     

Carbohydrate and Amino Acid Metabolism in the Ectomycorrhizal Ascomycete Sphaerosporella brunnea during Glucose Utilization : A C NMR Study

Nuclear magnetic resonance spectroscopy was utilized to study the metabolism of [1-¹³C]glucose in mycelia of the ectomycorrhizal ascomycete Sphaerosporella brunnea. The main purpose was to assess the biochemical pathways for the assimilation of glucose and to identify the compounds accumulated during glucose assimilation. The majority of the ¹³C label was incorporated into mannitol, while glycogen, trehalose and free amino acids were labeled to a much lesser extent. The high enrichment of the C1/C6 position of mannitol indicated that the polyol was formed via a direct route from absorbed glucose. Randomization of the ¹³C label was observed to occur in glucose and trehalose leading to the accumulation of [1,6-¹³C]trehalose and [1,6-¹³C]glucose. This suggests that the majority of the glucose carbon used to form trehalose was cycled through the metabolically active mannitol pool. The proportion of label entering the free amino acids represented 38% of the soluble ¹³C after 6 hours of continuous glucose labeling. Therefore, amino acid biosynthesis is an important sink of assimilated carbon. Carbon-13 was incorporated into [3-¹³C]alanine and [2-¹³C]-, [3-¹³C]-, and [4-¹³C]glutamate and glutamine. From the analysis of the intramolecular ¹³C enrichment of these amino acids, it is concluded that [3-¹³C]pyruvate, arising from [1-¹³C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase, and pyruvate carboxylase (or phosphoenolpyruvate carboxykinase). Intramolecular ¹³C labeling patterns of glutamate and glutamine were similar and are consistent with the operation of the Krebs cycle. There is strong evidence for (a) randomization of the label on C2 and C3 positions of oxaloacetate via malate dehydrogenase and fumarase, and (b) the dual biosynthetic and respiratory role of the citrate synthase, aconitase, and isocitrate dehydrogenase reactions. The high flux of carbon through the carboxylation (presumably pyruvate carboxylase) step indicates that CO₂ fixation is an important component of the carbon metabolism in S. brunnea, and it is likely that this anaplerotic role is particularly prevalent during NH₄⁺ assimilation. The most relevant information resulting from this investigation is (a) the occurrence of the mannitol cycle, (b) a large part of the trehalose pool is synthesized after the cycling of glucose-carbon through the mannitol cycle, and (c) pyruvate (or phosphoenolpyruvate) carboxylation plays an important role in the primary metabolism of glucose-fed mycelia.     

Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria

Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-¹³C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-¹³C]-, [2-¹³C]-, or [3-¹³C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.     

Elongation Pathway for alpha-Linolenic Acid Synthesis in Spinach Leaves: A Reexamination

Leaf slices from spinach exhibited considerable variation in their incorporation of [¹⁴C]bicarbonate and [1-¹⁴C]acetate into fatty acids. Reductive ozonolysis studies indicated that all the ¹⁴C-labeled fatty acids were synthesized de novo; no in vivo evidence was found for the previously proposed elongation of hexadecatrienoate to α-linolenate.     

ÈVIDENCE FOR THE COMPARTMENTATION OF GLUTAMATE METABOLISM IN ISOLATED RAT RETINA

—Glucose is a major precursor of glutamate and related amino acids in the retina of adult rats. ¹⁴C from labelled glucose appears to gain access to a large glutamate pool, and the resulting specific activity of glutamate labelled from glucose is always higher than that of glutamine or the other amino acids. Radioactive acetate appeared to label a small glutamate pool. The specific activity of glutamine labelled from acetate relative to that of glutamate was always greater than 1.0. Other precursors of the small glutamate pool were found to include glutamate, aspartate, GABA, serine, leucine and sodium bicarbonate. The level of radioactivity present in retinae incubated with [U-¹⁴C]glucose or [1-¹⁴C]sodium acetate was reduced in the presence of 10^?5m -ouabain. Under these conditions, the relative specific activity of glutamine labelled from [1-¹⁴C]sodium acetate was lowered, but it was raised when [U-¹⁴C]glucose was used as substrate. Ouabain also considerably reduced the synthesis of GABA from [1-¹⁴C]sodium acetate. In all cases ouabain caused a fall in the tissue levels of the amino acids. Aminooxyacetic acid (10^?4m ) almost completely abolished the labelling of GABA from both [U-¹⁴C]glucose and [1-¹⁴C]sodium acetate, while the RSA of glutamine labelled from the latter substrate was significantly increased. Aminooxyacetic acid raised the tissue concentration of glutamate, but caused a fall in the tissue concentrations of glutamine, aspartate and GABA. The results suggest that there are separate compartments for the metabolism of glutamate in retina and that these can be modified in different ways by different drugs.     

Metabolism of Hydroxylated and Fluorinated Benzoates by Syntrophus aciditrophicus and Detection of a Fluorodiene Metabolite

Transformations of 2-hydroxybenzoate and fluorobenzoate isomers were investigated in the strictly anaerobic Syntrophus aciditrophicus to gain insight into the initial steps of the metabolism of aromatic acids. 2-Hydroxybenzoate was metabolized to methane and acetate by S. aciditrophicus and Methanospirillum hungatei cocultures and reduced to cyclohexane carboxylate by pure cultures of S. aciditrophicus when grown in the presence of crotonate. Under both conditions, transient accumulation of benzoate but not phenol was observed, indicating that dehydroxylation occurred prior to ring reduction. Pure cultures of S. aciditrophicus reductively dehalogenated 3-fluorobenzoate with the stoichiometric accumulation of benzoate and fluorine. 3-Fluorobenzoate-degrading cultures produced a metabolite that had a fragmentation pattern almost identical to that of the trimethylsilyl (TMS) derivative of 3-fluorobenzoate but with a mass increase of 2 units. When cells were incubated with deuterated water, this metabolite had a mass increase of 3 or 4 units relative to the TMS derivative of 3-fluorobenzoate. ¹⁹F nuclear magnetic resonance spectroscopy (¹⁹F NMR) detected a metabolite in fluorobenzoate-degrading cultures with two double bonds, either 1-carboxyl-3-fluoro-2,6-cyclohexadiene or 1-carboxyl-3-fluoro-3,6-cyclohexadiene. The mass spectral and NMR data are consistent with the addition of two hydrogen or deuterium atoms to 3-fluorobenzoate, forming a 3-fluorocyclohexadiene metabolite. The production of a diene metabolite provides evidence that S. aciditrophicus contains dearomatizing reductase that uses two electrons to dearomatize the aromatic ring.     

Stereochemistry of the reactions catalyzed by chicken liver fatty acid synthase

The stereochemistry of the four partial reactions catalyzed by chicken liver fatty acid synthase that lead to the synthesis of palmitic acid has been determined. The reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by NADPH proceeds with the transfer of the pro-4S hydrogen of NADPH to form D-3-hydroxybutyryl-CoA. During the subsequent dehydration of D-3-hydroxybutyryl-CoA the pro-2S hydrogen and the 3-hydroxyl group are removed in a syn elimination to form crotonyl-CoA. Crotonyl-CoA is reduced to butyryl-CoA by NADPH, with the transfer of the pro-4R hydrogen of NADPH to the pro-3R position in butyryl-CoA and the transfer of a solvent hydrogen to the pro-2S position. The occurrence of the syn dehydration, when combined with the results of a previous study [ Sedgwick , B., & Cornforth , J. W. (1977) Eur. J. Biochem. 75, 465-479], implies that the condensation of the enzyme-bound malonyl moiety with the enzyme-bound saturated fatty acid to form a 3-keto intermediate proceeds with inversion at C-2 of the malonyl. The stereochemistry of the hydration was derived from an analysis of the spin-spin coupling constant of 3-hydroxy[2-2H]butyric acid benzylamides obtained from 3-hydroxy[2-2H]butyryl-CoA synthesized by fatty acid synthase. The elucidation of the stereochemistry of the reduction of crotonyl-CoA relied on the previously established stereochemistry of pork liver acyl-CoA dehydrogenase. The source of all 28 prochiral hydrogens of the palmitic acid synthesized by chicken liver fatty acid synthase was inferred from the results of this work.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

COMPARTMENTATION OF AMINO ACID METABOLISM IN THE RAT POSTERIOR PITUITARY

—Isolated rat posterior pituitary glands were incubated with [¹⁴C]glucose or [¹⁴C]acetate and the incorporation of radioactivity into several amino acids was followed. The results indicated that radioactivity was incorporated from [¹⁴C]glucose into a large pool of glutamate which appeared to be responsible for a large proportion of GABA synthesis in the gland. The specific activity of glutamine was always less than that of glutamate when [¹⁴C]glucose was the precursor employed, whereas [¹⁴C]acetate labelled a glutamate pool which had approximately the same specific activity as that of glutamine. The results are discussed with reference to the compartmentation of amino acid metabolism in the nervous system.     

Compartmentation of citric acid cycle metabolism in brain: effect of aminooxyacetic acid, ouabain and Ca2+ on the labelling of glutamate, glutamine, aspartate and gaba by [1-14C]acetate, [U-14C]glutamate and [U-14C]asparate

—(1) The effects of aminooxyacetic acid, ouabain and Ca²⁺ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium-[1-¹⁴C]acetate, l-[U-¹⁴C]glutamate and l-[U-¹⁴C]aspartate as tracer metabolites. (2) Aminooxyacetic acid (10^-3 m) inhibited the labelling of aspartate from [¹⁴C]acetate and [¹⁴C]glutamate, as well as the incorporation of label from [¹⁴C]aspartate into glutamate and glutamine. It also inhibited the labelling of GABA from all three radioactive precursors, as would be anticipated if there was inhibition of several transaminases as well as glutamate decarboxylase. The RSA of glutamine labelled from [1-¹⁴C]acetate was increased. This finding indicated that the glutamate pool which is utilized for glutamine formation is associated with glutamate dehydrogenase, and this enzyme appears to be related to the ‘synthetic tricarboxylic acid cycle’. AOAA exerted its major inhibitory effects on the citric acid‘energy cycle’with which transaminases are associated. (3) Ouabain (10^-5 m) inhibited the labelling of glutamine to a much greater extent than the labelling of glutamate from [1-¹⁴C]acetate. It also caused leakage of amino acids from the tissue into the medium. Its effect on the glutamate–glutamine system was interpreted to be a selective inhibition of the 'synthetic’citric acid cycle. (4) The omission of Ca²⁺ from the incubation medium was associated with formation of glutamine with RSA less than 1·0 when labelled from [U-¹⁴C]glutamate, [U-¹⁴C]aspartate and lower than normal when labelled from [1-¹⁴C]acetate.     

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

CO2 Incorporation and 4-Hydroxy-2-Methylbenzoic Acid Formation during Anaerobic Metabolism of m-Cresol by a Methanogenic Consortium

The metabolism of m-cresol by methanogenic cultures enriched from domestic sewage sludge was investigated. In the initial studies, bromoethanesulfonic acid was used to inhibit methane production. This led to the accumulation of 4.0 ± 0.8 mol of acetate per mol of m-cresol metabolized. These results suggested that CO₂ incorporation occurred because each molecule of m-cresol contained seven carbon atoms, whereas four molecules of acetate product contained a total of eight carbon atoms. To verify this, [¹⁴C]bicarbonate was added to bromoethanesulfonic acid-inhibited cultures, and those cultures yielded [¹⁴C]acetate. Of the label recovered as acetate, 89% was found in the carboxyl position. Similar cultures fed [methyl-¹⁴C]m-cresol yielded methyl-labeled acetate. A ¹⁴C-labeled transient intermediate was detected in cultures given either m-cresol and [¹⁴C]bicarbonate or bicarbonate and [methyl-¹⁴C]m-cresol. The intermediate was identified as 4-hydroxy-2-methylbenzoic acid. In addition, another metabolite was detected and identified as 2-methylbenzoic acid. This compound appeared to be produced only sporadically, and it accumulated in the medium, suggesting that the dehydroxylation of 4-hydroxy-2-methylbenzoic acid led to an apparent dead-end product.     

Neuron–Astrocyte Interactions,Pyruvate Carboxylation and the Pentose Phosphate Pathway in the Neonatal Rat Brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-¹³C]glucose and were sacrificed 30 min later. Brain extracts were analysed using ¹H- and ¹³C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-¹³C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-¹³C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-¹³C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.