acetyl-CoA Pathway Research Articles

Carbon Isotopic Signatures of Individual Archean Microfossils(?) from Western Australia

New types of carbonaceous filamentous microstructures have been identified in silica veins at two new localities in the ∼3.5 Ga North Pole area of Western Australia. Their carbon isotopic compositions were measured in situ by secondary-ion mass spectrometry. The carbonaceous filaments are ∼1μm wide, 10 to 100 μm long, and are permineralized in a fine-grained (∼1 μm) silica matrix. They are morphologically divided into three types (i.e., spiral, threadlike, and branched filaments). Their sizes and morphologies resemble modern and previously reported fossil bacteria. These similarities and their complex three-dimensional geometry suggest that they may represent morphologically preserved fossil bacteria. δ13C values of the carbonaceous filaments range from −42 to −32‰, which strongly suggest that they are composed of biologically fixed organic compounds, possibly via the reductive acetyl-CoA pathway or the Calvin cycle. This is consistent with the hypothesis that autotrophs already existed on the Archean Earth.

Clostridium scatologenes strain SL1 isolated as an acetogenic bacterium from acidic sediments.

A strictly anaerobic, H2-utilizing bacterium, strain SL1, was isolated from the sediment of an acidic coal mine pond. Cells of strain SL1 were sporulating, motile, long rods with a multilayer cell wall. Growth was observed at 5-35 degrees C and pH 3.9-7.0. Acetate was the sole end product of H2 utilization and was produced in stoichiometries indicative of an acetyl-CoA-pathway-dependent metabolism. Growth and substrate utilization also occurred with CO/CO2, vanillate, syringate, ferulate, ethanol, propanol, 1-butanol, glycerine, cellobiose, glucose, fructose, mannose, xylose, formate, lactate, pyruvate and gluconate. With most substrates, acetate was the main or sole product formed. Growth in the presence of H2/CO2 or CO/CO2 was difficult to maintain in laboratory cultures. Methoxyl, carboxyl and acrylate groups of various aromatic compounds were O-demethylated, decarboxylated and reduced, respectively. Small amounts of butyrate were produced during the fermentation of sugars. The acrylate group of ferulate was reduced. Nitrate, sulfate, thiosulfate, dimethylsulfoxide and Fe(III) were not utilized as electron acceptors. Analysis of the 16S rRNA gene sequence of strain SL1 demonstrated that it is closely related to Clostridium scatologenes (99.6% sequence similarity), an organism characterized as a fermentative anaerobe but not previously shown to be capable of acetogenic growth. Comparative experiments with C. scatologenes DSM 757T demonstrated that it utilized H2/CO2 (negligible growth), CO/CO2 (negligible growth), formate, ethanol and aromatic compounds according to stoichiometries indicative of the acetyl-CoA pathway. CO dehydrogenase, formate dehydrogenase and hydrogenase activities were present in both strain SL1 and C. scatologenes DSM 757T. These results indicate that (i) sediments of acidic coal mine ponds harbour acetogens and (ii) C. scatologenes is an acetogen that tends to lose its capacity to grow acetogenically under H2/CO2 or CO/CO2 after prolonged laboratory cultivation.

Carbon isotopic composition of individual Precambrian microfossils.

Ion microprobe measurements of carbon isotope ratios were made in 30 specimens representing six fossil genera of microorganisms petrified in stromatolitic chert from the approximately 850 Ma Bitter Springs Formation, Australia, and the approximately 2100 Ma Gunflint Formation, Canada. The delta 13C(PDB) values from individual microfossils of the Bitter Springs Formation ranged from -21.3 +/- 1.7% to -31.9 +/- 1.2% and the delta 13C(PDB) values from microfossils of the Gunflint Formation ranged from -32.4 +/- 0.7% to -45.4 +/- 1.2%. With the exception of two highly 13C-depleted Gunflint microfossils, the results generally yield values consistent with carbon fixation via either the Calvin cycle or the acetyl-CoA pathway. However, the isotopic results are not consistent with the degree of fractionation expected from either the 3-hydroxypropionate cycle or the reductive tricarboxylic acid cycle, suggesting that the microfossils studied did not use either of these pathways for carbon fixation. The morphologies of the microfossils suggest an affinity to the cyanobacteria, and our carbon isotopic data are consistent with this assignment.

Mechanism of transfer of the methyl group from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum: a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis.

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5736-5745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

Presence of acetyl coenzyme A (CoA) carboxylase and propionyl-CoA carboxylase in autotrophic Crenarchaeota and indication for operation of a 3-hydroxypropionate cycle in autotrophic carbon fixation.

The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

Flavonoid Biosynthetic Pathway and Cereal Defence Response: An Emerging Trend in Crop Biotechnoloy

The flavonoid pathway, derived from the phenylpropanoid and acetyl CoA and malonyl CoA pathways, gives rise to a diverse array of compounds such as isoflavonoids, anthocyanins, proanthocyanidins, etc. that are attributed with a multitude of biological functions. There is an increasing evidence on the role of flavonoids in defence response in many plant species though the exact mechanism is yet to be defined unequivocally. On the contrary, there is very little information on the role of flavonoids in cereal defence response. A novel strategy to improve disease resistance in cereals is by the engineering of the flavonoid pathway for enhancement of specific flavonoids in selected tissue under pathogen attack. Recent developments in molecular biology and genetics of flavonoid pathway in several plants would allow one to test its potential in improving disease resistance. Further, the powerful molecular techniques and reproducible transformation protocols would enable the manipulation of the flavonoid pathway in cereals. Transferring allied pathways, or a part of pathways to target plants seem to be within the reach of many groups. It is therefore, time for a second look at this secondary pathway particularly in the context of disease resistance phenomenon.

Acetogenic bacteria: what are the in situ consequences of their diverse metabolic versatilities?

The four decades of the now classic studies by Harland G. Wood and Lars G. Ljungdahl lead to the resolution of the autotrophic acetyl-CoA 'Wood/Ljungdahl' pathway of acetogenesis. This pathway is the hallmark of acetogens, but is also used by other bacteria, including methanogens and sulfate-reducing bacteria, for both catabolic and anabolic purposes. Thus, the pathway is wide spread in nature and plays an important role in the global turnover of carbon. Because most historical studies with acetogens focused on the biochemistry of the acetyl-CoA pathway, the metabolic diversity and ecology of acetogens remained largely unexplored for many years. Although acetogens were initially conceived to be a somewhat obscure bacteriological group with limited metabolic capabilities, it is now clear that acctogens are arguably the most metabolically diverse group of obligate anaerobes characterized to date. Their anaerobic metabolic arsenal includes the capacity to oxidize diverse substrates, including aromatic, C1, C2, and halogenated compounds, and engage a large number of alternative energy-conserving, terminal electron-accepting processes, including classic fermentations and the dissimilation of inorganic nitrogen. In this regard, one might consider acetogens on a collective basis as the pseudomonads of obligate anaerobes. By virtue of their diverse metabolic talents, acetogens can be found in essentially all habitats. This review evaluates the metabolic versatilities of acetogens relative to both the engagement (regulation) of the acetyl-CoA pathway and the ecological roles likely played by this bacteriogical group.

Effect of nitrate on the autotrophic metabolism of the acetogens Clostridium thermoautotrophicum and Clostridium thermoaceticum.

Although nitrate stimulated the capacity of Clostridium thermoautotrophicum and Clostridium thermoaceticum to oxidize (utilize) substrates under heterotrophic conditions, it inhibited autotrophic H2-CO2-dependent growth. Under basal medium conditions, nitrate was also inhibitory to the use of one-carbon substrates (i.e., CO, formate, methanol, or the O-methyl groups of vanillate or syringate) as sole carbon energy sources. This inhibitory effect of nitrate was bypassed when both O-methyl groups and CO were provided concomitantly; H2-CO2 did not replace CO. These results indicated that nitrate blocked the reduction of CO2 to the methyl and carbonyl levels. On the basis of the inability of acetogenic cells (i.e., cells cultivated without nitrate) to consume or reduce nitrate in resting-cell assays, the capacity to dissimilate nitrate was not constitutive. Nitrate had no appreciable effect on the specific activities of enzymes central to the acetyl-coenzyme A (CoA) pathway. However, membranes obtained from cells cultivated under nitrate-dissimilating conditions were deficient in the b-type cytochrome that was typical of membranes from acetogenic cells, i.e., cells dependent upon the synthesis of acetate for the conservation of energy. Collectively, these findings indicated that (i) C. thermoautotrophicum and C. thermoaceticum cannot engage the carbon-fixing capacities of the acetyl-CoA pathway in the presence of nitrate and (ii) the nitrate block on the acetyl-CoA pathway occurs via an alteration in electron transport.

Bidirectional usage of ferulate by the acetogen Peptostreptococcus productus U-1: CO2 and aromatic acrylate groups as competing electron acceptors

The influence of CO2 on the ability of Peptostreptococcus) productus U-1 (ATCC 35244) to use an aromatic acrylate group as an energy-conserving electron acceptor during O-methyl-dependent growth was examined. Ferulate (a methoxylated phenylacrylate), unlike hydroferulate (a methoxylated phenylpropionate),supported growth under CO2-limited conditions. Two phases occurred during ferulate utilization in CO2-limited cultures. In phase I (maximum growth), O-methyl-derived reductant was coupled mainly to acrylate group reduction, and acetate synthesis (CO2 as reductant sink) was minimal. In phase II, acetate synthesis increased, but cell yields in this phase were much less than in phase I. In CO2-enriched cultures, distinct phases were not observed; reductant was coupled equally to CO2 and acrylate group reduction. Under CO2-enriched conditions, O-methyl and acrylate groups were incompletely metabolized, and molar growth yields were significantly lower compared to CO2-limited conditions. Resting cell studies indicated that O-demethylase and aromatic acrylate oxidoreductase activities were induced by ferulate. These findings demonstrated that P. productus U-1 can use the aromatic acrylate oxidoreductase system as a sole, energy-conserving, electron-accepting process, but is not able to prevent the simultaneous use of the bioenergetically less favourable acetyl-CoA pathway during O-methyl-dependent growth.

A conformational change in the methyltransferase from Clostridium thermoaceticum facilitates the methyl transfer from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein in the Acetyl-CoA pathway.

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the methylation of a corrinoid/iron-sulfur protein (C/Fe-SP) by the N5 methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate). This is an important reaction in the reductive acetyl-CoA pathway. The forward and reverse reactions of MeTr have a pH dependence that appears to reflect protonation of a group on the protein [Zhao, S., Roberts, D. L., & Ragsdale, S. W. (1995) Biochemistry 34, 15075-15083]. In the work reported here, fluorescence and rapid reaction kinetics were used to demonstrate that this protonation elicits a rate-limiting conformational change. As the pH was lowered, the emission maximum for intrinsic tryptophan fluorescence underwent a red shift (pK(a) = 5.4) and the emission intensity increased (pK(a) = 5.1). The extrinsic fluorescence probe, 4,4'-bis-1-phenylamino-8-napthalenesulfonate (bis-ANS) was used to report on the conformational change. The bis-ANS fluorescence was strongly enhanced upon binding MeTr. As the pH was decreased, the fluorescence was further enhanced and the emission maximum underwent a 14 nm blue shift (pK(a) = 5.0). By stopped-flow fluorescence studies, it was shown that these fluorescence changes occur at rates similar to the k(cat) for the MeTr reaction and thus reflect catalytically competent events. The combined results indicate that CH3-H4folate binds to a hydrophobic region in MeTr that includes a tryptophan residue(s). MeTr undergoes a pH-dependent conformational change that exposes this region to solvent and facilitates substrate binding.

Acetogenic coccoid spore-forming bacteria isolated from the rumen

Thirteen strains of a new acetogenic bacterium were isolated from the rumen contents of lambs, llamas and bisons. This paper is the first report of Gram-positive coccoid spore-forming bacteria occurring in chains and able to use H 2 + CO 2 as energy source and produce acetate from this gas mixture. One of them, chosen as the reference strain for its efficiency in utilizing H 2 CO 2 likely via the acetyl-CoA pathway, was characterized in detail. The G+C ratio of the DNA of the organism was 46.5 mol%. The temperature and pH optimum were 37°–40°C and 6.3–6.8, respectively. Numerous organic substrates including some o-methylate aromatic compounds were used heterotrophically. The full 16S rRNA gene sequence was determined. The phylogeny, physiology, morphology and numerous features described here are sufficiently different from those of any bacteria described today to justify the definition of a new species. The name “New acetogenic bacterium” is temporarily proposed, awaiting a future taxonomic revision of the genus Clostridium.

Growth on methanol and conversion of methoxylated aromatic substrates by Desulfotomaculum orientis in the presence and absence of sulfate

A three-membered microbial community that completely metabolizes syringic acid to methane and carbon dioxide was originally enriched from anoxic sediment of the lake Zürich. From this enrichment a sulfate- reducing, demethylating, spore-forming organism, classified as Desulfotomaculum orientis, was isolated. It cleaves the phenylether bonds of methyxylated aromatic compounds, producing the corresponding hydroxylated derivatives. The organism ferments the carbon of methoxy-groups as well as methanol to acetate using carbon dioxide as electron acceptor. In the presence of sulfate, methoxy carbon and methanol are completely oxidized to CO 2 coupled to the formation of hydrogen sulfide. Growth yields under fermenting conditions are 4.0 to 6.4 g of dry cell mass per mol of methanol or methoxy-carbon consumed. Under sulfate reducing conditions the corresponding values are 4.8 to 8.2 g for methanol and methoxy-carbon. The acetyl-CoA pathway is proposed as the main metabolic route for assimilation and dissimilation of these C 1-substrates. Acetate added to the culture medium cannot be utilized and it is not needed to promote growth in solely C-1 substrate containing media neither under sulfate-reducing nor under fermenting conditions. The ability of Desulfotomaculum orientis to endure unfavorable environmental conditions through sporulation, its versatility with regard to substrate utilization and its metabolic flexibility make it well adapted to be a member of transient anoxic fresh water sediment communities.

Mechanistic studies of the methyltransferase from Clostridium thermoaceticum: origin of the pH dependence of the methyl group transfer from methyltetrahydrofolate to the corrinoid/iron-sulfur protein.

A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP). The methylcobamide product is the first in a series of enzyme-bound organometallic intermediates in the acetyl-CoA pathway of anaerobic CO2 fixation. The mechanisms of the forward and reverse reactions with CH3-H4folate and either the C/Fe-SP or vitamin B12 as substrates were studied by steady-state and pre-steady-state kinetics. This ability to effectively utilize free cobalamin as well as the C/Fe-SP in the transmethylation appears to explain why [14C]methylcobyric acid was found as a product of labeling C. thermoaceticum cells with 14CO2 [Ljungdahl, L. G., Irion, E., & Wood, H. G. (1965) Biochemistry 4, 2771-2780]. Stopped-flow experiments indicate that the Co(I)-C/Fe-SP performs a direct SN2 displacement of the methyl group of CH3-H4folate to form H4folate and methyl-Co(III). The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pKa approximately 5.5). Similar pH profiles were obtained by steady-state kinetics. The kcat/Km values for the C/Fe-SP and CH3-H4folate in the forward direction and for the methylated C/Fe-SP and H4folate in the reverse direction increased as the pH was lowered (pKa approximately 5.3). A different pH profile was obtained with free cobalamin as the substrate; the kcat/Km for CH3-H4folate and cobalamin (forward reaction) increased (pKa approximately 7.0) and the kcat/Km for H4folate and methylcobalamin (reverse reaction) decreased (pKa approximately 5.3) as the pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)

Enumeration of H2-utilizing methanogenic archaea, acetogenic and sulfate-reducing bacteria from human feces

Fecal specimens from 19 healthy humans were used to enumerate H2-utilizing microbial populations of methanogenic archaea (MA), acetogenic bacteria (AB) and sulfate-reducing bacteria (SRB). Eight subjects were methane (CH4) excretors (CH4 +) and 11 non CH4-excretors (CH4 −), based on breath methane concentrations. The mean ± S.E. of the logarithm of MA per gram wet weight feces were 8.8 ± 0.21 and 2.6 ± 0.39 for CH4+ and CH4−, respectively (P < 0.001). SRB counts were 7.1 ± 0.43 and 7.3 ± 0.39, respectively (NS), while counts of AB were 4.6 ± 0.75 and 6.6 ± 0.38, respectively (P < 0.02). Counts of AB were negatively correlated with counts of MA (r = −0.53; P < 0.05). These results confirm the potential importance of AB in the human colon, especially for CH4 — subjects, and suggest that a much greater competitive interrelation occurs in the human colon between MA and AB than between the former and SRB. We further report on the isolation of representatives of the dominant H2CO2 acetogenic population. Three strains from two CH4 — subjects were characterized from 10−5-10−7 dilutions. They all consumed H2CO2 and several carbohydrates to produce acetate as the sole metabolite. Phenotypically related to the species Peptostreptococcus productus, the strains used H2CO2 via the acetyl-CoA pathway.

Enumeration of H 2-utilizing methanogenic archaea, acetogenic and sulfate-reducing bacteria from human feces

Fecal specimens from 19 healthy humans were used to enumerate H 2-utilizing microbial populations of methanogenic archaea (MA), acetogenic bacteria (AB) and sulfate-reducing bacteria (SRB). Eight subjects were methane (CH 4) excretors (CH 4 +) and 11 non CH 4-excretors (CH 4 −), based on breath methane concentrations. The mean ± S.E. of the logarithm of MA per gram wet weight feces were 8.8 ± 0.21 and 2.6 ± 0.39 for CH 4+ and CH 4−, respectively ( P < 0.001). SRB counts were 7.1 ± 0.43 and 7.3 ± 0.39, respectively (NS), while counts of AB were 4.6 ± 0.75 and 6.6 ± 0.38, respectively ( P < 0.02). Counts of AB were negatively correlated with counts of MA (r = −0.53; P < 0.05). These results confirm the potential importance of AB in the human colon, especially for CH 4 — subjects, and suggest that a much greater competitive interrelation occurs in the human colon between MA and AB than between the former and SRB. We further report on the isolation of representatives of the dominant H 2 CO 2 acetogenic population. Three strains from two CH 4 — subjects were characterized from 10 −5-10 −7 dilutions. They all consumed H 2 CO 2 and several carbohydrates to produce acetate as the sole metabolite. Phenotypically related to the species Peptostreptococcus productus, the strains used H 2 CO 2 via the acetyl-CoA pathway.

Anaerobic pathway for conversion of the methyl group of aromatic methyl ethers to acetic acid by Clostridium thermoaceticum.

Clostridium thermoaceticum and other anaerobic acetogenic bacteria can utilize the methyl group of aromatic methyl ethers as a carbon and energy source. It has been unclear what pathway is used to metabolize this methyl group. In the work reported here, the pathway was established by identifying and quantitating the substrates, stable intermediates, and products of O-demethylation of syringic acid. By measuring the dependence of the O-demethylation reaction on purified enzymes of the acetyl-CoA pathway, it was established that CO dehydrogenase, the corrinoid/iron-sulfur protein, and methyltransferase all were required for acetyl-CoA formation. By 13C-NMR spectroscopy it was shown that the O-demethylase from C. thermoaceticum converts the methyl group of syringate to methyltetrahydrofolate (CH3-H4folate). When the reaction was conducted in the presence of CO, H2, or titanium(III), or in the absence of any electron donor, the rate of demethylation of syringic acid at pH 7.2 was approximately 15 nmol min-1 mg-1. In the absence of CO, CH3-H4folate accumulated as a stable product. When CO was added, 13CH3-H4folate was converted to [2-13C]acetyl-CoA, [2-13C]acetyl phosphate, and [2-13C]acetate. Therefore, the acetogenic O-demethylase uses H4folate as acceptor of the methyl group of phenyl methyl ethers and catalyzes the formation of CH3-H4folate. The pathway of conversion of CH3-H4folate, CO, and CoA to acetyl-CoA has been studied previously. Methyltransferase catalyzes the reaction of CH3-H4folate with the corrinoid/iron-sulfur protein to form a methylcobalt species. The nickel/iron-sulfur enzyme CO dehydrogenase then catalyzes the final steps in the formation of acetyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)

Competition of CO 2 and Acetate as Substrates for Fatty Acid Synthesis in Immature Chloroplasts of Barley Seedlings

Summary Immature chloroplasts located at the base of barley leaves near the meristematic zone incorporated 14 C from NaH 14 CO 3 into fatty acids at rates resembling (or some times exceeding) rates from [2- 14 C]acetate. Non-labeled acetate added at concentrations comparable to NaH 14 CO 3 did not affect the 14C-incorporation. In immature chloroplasts the chloroplastic pyruvate dehydrogenase complex (chIp PDC), which is the terminal enzyme of the chloroplast 3-phosphoglycerate ➙ acetyl-CoA pathway (Hoppe et al. 1993), was considerably more active than the acetyl-CoA synthetase (ACS). During chloroplast development, the carbon flow through this pathway decreased. At the same time, ACS activity increased and met the requirement for an effective acetate utilization for fatty acid synthesis in mature chloroplasts.

Acetogenesis and ATP synthesis inAcetobacterium itwoodiiare coupled via a transmembrane primary sodium ion gradient

In cell suspensions of Acetobacterium woodii the acetyl-CoA pathway is coupled to net ATP formation. Acetate formation as well as ATP synthesis and the generation of a transmembrane sodium ion gradient are not inhibited by protonophores but by sodium ionophores. Acetogenesis from CO or formaldehyde + CO as catalyzed by inverted vesicles is coupled to sodium ion uptake. Both processes are not inhibited by protonophores but by sodium ionophores. These experiments are in accordance with the presence of a primary sodium ion pump connected to the acetyl-CoA pathway which enables the cells to synthesize net ATP by means of a ΔμNa+ in concert with a Na+-translocating ATPase.

Growth inhibition and pyruvate overflow during glucose metabolism of Eubactevium limosum are related to a limited capacity to reassimilate CO2 by the acetyl-CoA pathway

SUMMARY: The growth characteristics of Eubactevium limosum, an anaerobic acetogen, have been described kinetically for fermentation of glucose. A short-lived exponential growth phase was associated with a homoacetogenic fermentation in which all catabolic CO2 liberated was reassimilated via the acetyl-CoA pathway. During this phase no additional products were observed. A major shift in the metabolism leading to a mixed-acid fermentation pattern (acetate and butyrate) occurred coincident with the onset of the linear growth phase. Significantly diminished yields of acetate in this decelerating growth period were attributed to the limited capacity of the enzymes responsible for reductive generation of methyl donor within the acetyl-CoA pathway: CO2 and H2 accumulated in the gas phase. Pyruvate overflow (and to a lesser extent lactate, glycerate and carbon monoxide accumulation) were also observed, though pyruvate-ferredoxin oxidoreductase specific activity remained constant. In all experiments specific biomass formation rates were directly related to the rate of acetyl-CoA formation. The exponential growth phase was prolonged and the production of all compounds other than acetate was diminished when the medium was supplemented with tungsten, suggesting that formate dehydrogenase may be the enzyme limiting the correct functioning of the acetyl-CoA pathway.

The plastidic 3-phosphoglycerate ? acetyl-CoA pathway in barley leaves and its involvement in the synthesis of amino acids, plastidic isoprenoids and fatty acids during chloroplast development

Earlier studies on the synthesis of C3-derived amino acids, plastidic isoprenoids and fatty acids from CO2 by isolated chloroplasts in the light indicate the presence of a complete, but low-capacity, chloroplast (chlp) 3-phosphoglycerate → acetyl-CoA pathway which is predominantely active in immature (developing) chloroplasts (A. Heintze et al., 1990, Plant Physiol. 93, 1121–1127). In this paper, we demonstrate the activity of the enzymes involved i.e. chlp phosphoglycerate mutase, chlp enolase, chlp pyruvate kinase and chlp pyruvate-dehydrogenase complex (PDC), in the stroma of purified barley (Hordeum sativum L.) chloroplasts of different developmental stages. The chlp phosphoglycerate mutase was partially purified for the first time. The activities of the enzymes of this chlp pathway (except PDC) were about a magnitude lower than those of the cytosolic enzymes. The chlp PDC of barley was more active than that of spinach. The apparent K m values of the enzymes of this pathway were about 100 μM or lower except for the chlp phosphoglycerate mutase which had a K m of 1.6–1.8 mM for 3-phospho-d-glycerate. Interestingly, no appreciable change in the activity of these enzymes was observed during maturation of the chloroplasts. In contrast, the activity of the reversible NADP+-glyceraldehyde 3-phosphate dehydrogenase increased about five times (from 140 to 590 nkat per g leaf dry weight). The following hypothesis is put forward to explain the regulation of carbon metabolism during chloroplast development: 3-phospho-d-glycerate is withdrawn from a common pool by the actions of 3-phosphoglycerate kinase and NADP+-glyceraldehyde-3-phosphate dehydrogenase, the activity of which increases considerably during maturation of chloroplasts. This leads to an insufficient supply of 3-phospho-glycerate for the chlp phosphoglycerate mutase, which has a low affinity for its substrate.