Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate

Cell suspensions of Methanosarcina barkeri (strain Fusaro) grown on acetate were found to catalyze the formation of methane and CO₂ from acetate (30–40 nmol/min·mg protein) and an isotopic exchange between the carboxyl group of acetate and ¹⁴CO₂ (30–40 nmol/min·mg protein). An isotopic exchange between [¹⁴C]-formate and acetate was not observed. Cells grown on methanol mediated neither methane formation from acetate nor the exchange reactions. The data indicate that the isotopic exchange between CO₂ and the carboxyl group of acetate is a partial reaction of methanogenesis from acetate. Both reactions were completely inhibited by low concentrations of cyanide (20 M) or of hydrogen (0.5% in the gas phase). Methane formation from acetate was also completely inhibited by low concentrations of carbon monoxide (0.2% in the gas phase) whereas only significantly higher concentrations of CO had an effect on the exchange reaction. In the concentration range tested KCN, H₂ and CO had no effect on methane formation from methanol or from H₂ and CO₂; however, cyanide (20 M) also affected methane formation from CO. The results are discussed with respect to proposed mechanisms of methane and CO₂ formation from acetate.     

Mechanism of acetate synthesis from CO2 by Clostridium acidiurici.

Total synthesis of acetate from CO2 by Clostridium acidiurici during fermentations of hypoxanthine has been shown to involve synthesis of glycine from methylenetetrahydrofolate, CO2, and NH3. The glycine is converted to serine by the addition of methylenetetrahydrofolate, and the resulting serine is converted to pyruvate, which is decarboxylated to form acetate. Since CO2 is converted to methylenetetrahydrofolate, both carbons of the acetate are derived from CO2. The evidence supporting this pathway is based on (i) the demonstration that glycine decarboxylase is present in C. acidiurici, (ii) the fact that glycine is synthesized by crude extracts at a rate which is rapid enough to account for the in vivo synthesis of acetate from CO2, (iii) the fact that methylenetetrahydrofolate is an intermediate in the formation of both carbons of acetate from CO2, and (iv) the fact that the alpha carbon of glycine is the source of the carboxyl group of acetate. Evidence is presented that this synthesis of acetate does not involve carboxylation of a methyl corrinoid enzyme such as occurs in Clostridium thermoaceticum and Clostridium formicoaceticum. Thus, there are two different mechanisms for the total synthesis of acetate from CO2 by clostridia.     

Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum.

The catabolism of sodium formate to acetate and carbon dioxide by the anaerobic acetogen Butyribacterium methylotrophicum was analyzed by fermentation time course and 13C nuclear magnetic resonance studies. Significant hydrogen production and consumption fluxes were observed during formate catabolism but not during the catabolism of formate plus CO. In the latter case, formate and CO were simultaneously consumed and label distribution studies with mixtures of 13C-labeled CO and formate demonstrated their preferential incorporation into the acetate carboxyl and methyl groups, respectively. Hydrogen consumption was inhibited by CO when both were present, whereas hydrogen and formate were simultaneously consumed when CO2 was supplied. Carbon dioxide was required for the conversion of CO to acetate, but a similar need was not observed when methanol plus CO or formate plus CO was present. These analyses indicate a bifurcated single-carbon catabolic pathway in which CO2 is the sole single-carbon compound that directly supplies the carbonyl and methyl group synthesis pathways leading to the formation of acetyl coenzyme A, the primary reduced product. We discuss causes for the reported inability of B. methylotrophicum to use formate as a sole substrate.     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans oxidizes acetate to CO₂ with sulfate. This organism metabolizes acetate via a pathway in which C₁ units rather than tri- and dicarboxylic acids are intermediates. We report here that cell extracts of D. acetoxidans catalyzed an exchange between CO₂ and the carboxyl group of acetate at a rate of 90 nmol · min^-1 · mg^-1 protein which is sufficient to account for the in vivo acetate oxidation rate of 250 nmol · min^-1 · mg^-1 protein. The reaction was strictly dependent on both ATP and coenzyme A. The extracts contain high activities of acetate kinase (6.3 U · mg^-1 protein) and phosphotransacetylase (60 U · mg^-1 protein). These findings indicate that acetyl-CoA rather than acetyl-phosphate or acetate is the substrate of the carbon-carbon cleavage activity. Exchange was only observed in the presence of strong reducing agents such as Ti³⁺. Interestingly, the cell extracts also catalyzed the reduction of CO₂ to CO with Ti³⁺ as electron donor (120 nmol · min^-1 · mg^-1 protein). Carbon monoxide dehydrogenase and other oxidoreductases involved in acetate oxidation were found to be partially associated with the membrane fraction suggesting a membrane localization of these enzymes.Abbreviations MOPS Morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine - DTT d,l-1,4-Dithiothreitol - DMN 2,3-Dimethyl-1,4-naphthoquinone - MV_OX Methyl viologen, oxidized - APS Adenosinephosphosulfate - SRB Sulfate reducing bacteria - U mol product formed per min     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO₂ via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K _m; 1 U·mg^-1=1 mol·min^-1·mg protein^-1 at 37°C): Acetate kinase (6.3 U·mg^-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg^-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg^-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg^-1, 0.06 mM CH₃–FH₄); methylenetetrahydrofolate dehydrogenase (3.6 U·mg^-1, 0.9 mM NAD, 0.1 mM CH₂=FH₄); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg^-1); formyltetrahydrofolate synthetase (3 U·mg^-1, 1.4 mM FH₄, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg^-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg^-1.Non-standard abbreviations FH₄ Tetrahydrofolate - CHO-FH₄ N¹⁰-formyltetrahydrofolate - CHFH₄ N⁵,N¹⁰-methenyltetrahydrofolate - CH₂=FH₄ N⁵,N¹⁰-methylenetetrahydrofolate - CH₃–FH₄ N⁵-methyltetrahydrofolate - MOPS morpholinopropane sulfonic acid - DTT d,l-1,4-dithiothreitol - TRIS tris-(hydroxymethyl)-aminomethane - Ap₅A p¹,P⁵-di(adenosine-5)pentaphosphate - MV methyl viologen     

Recovery of labeled CO2 from acetate in severely burned children

The purpose of this study was to determine the fractional recovery rate of labeled CO(2) in the breath of severely burned children. This information is needed to perform tracer studies of substrate oxidation using carbon-labeled fatty acids. Nine children, ages 4-14 yr with massive burns participated in the study. All experiments were performed 7 days post burn after an overnight fast. A primed (60 micromol/kg), constant (2.0 micromol.kg(-1).min(-1)) infusion of [1,2-(13)C]acetate was given during a 4-h basal period and during a 4-h hyperinsulinemic euglycemic clamp. A priming dose (150 micromol/kg) of NaH(13)CO(3) was given at the beginning of the study. Breath samples were collected every 10 min during the last 40 min of each period. Indirect calorimetry was performed during the last 30 min of each period. The isotopic enrichment of (13)CO(2) was determined by isotope ratio-mass spectrometry, and total CO(2) excretion was measured by indirect calorimetry. The fractional recovery of acetate label was 0.89 +/- 0.05 and 0.88 +/- 0.04 during the basal state and clamp, respectively. We conclude that the fractional recovery of labeled acetate in severely burned children is approximately three times the recovery of a nonburned adult and similar to the value in exercising adults. The high recovery rate reflects the rapid turnover of the TCA cycle in burned children relative to the rate of exchange reactions. Minimal correction of expired CO(2) data is needed in this circumstance to quantify fatty acid oxidation using (13)C-labeled fatty acids.     

Fatty acid biosynthesis from acetate and CO2 in the developing soybean cotyledon

Developing soybean cotyledons rapidly incorporated acetate intofatty acids and water soluble constituents. Oleic acid was thefirst fatty acid to be detected with ¹⁴C and the ¹⁴C distributionpattern with time was consistent with its being the precursorof linoleic and linolenic acids. Palmitic or stearic acid didnot appear to be the precursor of oleic acid but appeared tobeformed parallel to it. The cotyledons did fix ¹⁴CO₂ by eitherdark or light fixation reactions but little ¹⁴C was incorporatedinto lipids. ¹Presented in part at the Midwest Section of the American Societyof Plant Physiologist Meeting in Columbia, Missouri, June 1970 (Received November 27, 1970; )     

Biosynthesis of monoterpenes in plants from 14C-labelled acetate and CO2

Degradation of (+)-isothujone biosynthesized by Tanacetum vulgare or Thuja plicata from acetate-[1-¹⁴C], -[2-¹⁴C] and -[2-³H₃] or from CO₂-[14C] at physiological concentration revealed a pattern of asymmetric labelling whereby tracer predominantly (72–98% resided in that part of the skeleton derived from IPP. This is similar to the patterns previously obtained for uptake of MVA-[2-¹⁴C] but differed from those reported in other species with acetate-[¹⁴C] as precursor. Within the IPP-derived moiety the 3 parts derived from acetate units were not equivalently labelled. Partial degradations of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium after uptake of ¹⁴C-labelled acetate or CO₂ showed that the C-2 units of the skeletons of these monoterpenes were also labelled to widely differing extents and these patterns persisted over a range of feeding and seasonal conditions. These results suggest that metabolic pools of acetyl-CoA and/or acetoacetyl-CoA exist in these plants. The general occurrence of such pools and the consequent nonequivalent labelling patterns in secondary metabolism could invalidate biosynthetic conclusions drawn from partial degradations of labelled natural products.     

Enumeration of bacteria forming acetate from H2 and CO2 in anaerobic habitats

A method has been worked out that allows the detection and isolation of bacteria fermenting molecular hydrogen and carbon dioxide to acetic acid.The ratio of methanogenic to acetogenic bacteria in sludge and lake sediment samples has been found to be approximately 100 to 1. Acetogenic bacteria could not be detected in rumen samples.     

Augmentation of CO2 drives by chlormadinone acetate, a synthetic progesterone

We studied 10 male subjects who were administered chlormadinone acetate (CMA), a potent synthetic progesterone, to clarify the physiological basis of its respiratory effects. Arterial blood gas tension, resting ventilation, and respiratory drive assessed by ventilatory and occlusion pressure response to CO2 with and without inspiratory flow-resistive loading were measured before and 4 wk after CMA administration. In all subjects, arterial PCO2 decreased significantly by 5.7 +/- 0.6 (SE) Torr with an increase in minute ventilation by 1.8 +/- 0.6 l X min-1, whereas no significant changes were seen in O2 uptake. During unloaded conditions, both slopes of occlusion pressure and ventilatory response to CO2 increased, being statistically significant in the former but showing nonsignificant trends in the latter. Furthermore, inspiratory flow-resistive loading (16 cmH2O X l(-1) X s) increased both slopes more markedly after CMA. The magnitudes of load compensation, assessed by the ratio of loaded to unloaded slope of the occlusion pressure response curve, were increased significantly. We concluded CMA is a potent respiratory stimulant that increases the CO2 chemosensitivity and neuromechanical drives in the load-compensation mechanism.     

Levels of enzymes involved in the synthesis of acetate from CO2 in Clostridium thermoautotrophicum

The acetogenic bacterium Clostridium thermoautotrophicum, grown on methanol, glucose, or CO2-H2, contained high levels of corrinoids, formate dehydrogenase, tetrahydrofolate enzymes, carbon monoxide dehydrogenase, and hydrogenase. Cell-free extracts catalyzed pyruvate-dependent formation of acetate from methyltetrahydrofolate. These results suggest that C. thermoautotrophicum synthesizes acetate from CO2 via a formate-tetrahydrofolate-corrinoid pathway.