Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus (original) (raw)
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Journal of Bacteriology, 2001
In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO(2) molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-(13)C(2)]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C(1) units derived therefrom and not into other cell compounds.
Evidence That Carbon Monoxide Is an Obligatory Intermediate in Anaerobic Acetyl-CoA Synthesis †
Biochemistry, 1996
Carbon monoxide is produced by several biological reactions. It is proposed to act as an intracellular signaling molecule and can serve as the carbon and electron source for certain bacteria. Direct evidence for a new biological role for CO is presented here. The results strongly indicate that CO is produced as an obligatory intermediate during growth of the acetogenic bacterium Clostridium thermoaceticum on glucose, H 2 /CO 2 , or aromatic carboxylic acids. Our results are consistent with earlier hypotheses of the intermediacy of CO during growth of acetogenic bacteria on CO 2 and hexoses [Diekert, G., & Ritter, M. (1983) FEMS Microbiol. Lett. 17, 299-302] and methanogenic Archaea on CO 2 [Stupperich, E., Hammel, K. E., Fuchs, G., & Thauer, R. K. (1983) FEBS Lett. 152, 21-23]. Therefore, CO production is a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis. The carbonyl group of acetyl-CoA is shown to be formed from the carboxyl group of pyruvate by the following steps. (i) Pyruvate undergoes decarboxylation by pyruvate:ferredoxin oxidoreductase to form acetyl-CoA and CO 2 . (ii) CO 2 is reduced to CO by the CODH site of the bifunctional enzyme CO dehydrogenase/acetyl-CoA synthase (CODH/ACS). (iii) CO generated in situ combines with the ACS active site to form a paramagnetic adduct that has been called the NiFeC species, and (iv) the bound carbonyl group combines with a bound methyl group and CoA to generate acetyl-CoA. To our knowledge, this paper represents the first demonstration of a pathway in which CO is produced and then used as a metabolic intermediate.
European Journal of Biochemistry, 1992
The unresolved autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4-13C1]succinate, and the 13C pattern in cell constituents was determined by 1H- and 13C-NMR spectroscopy of purified amino acids and other cell constituents. In both organisms succinate contributed to less than 10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate. The 13C-labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4-13C1]succinate was partially converted to double-labelled [3,4-13C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed. The 13C-labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3-hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3-hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl-CoA carboxylase and propionyl-CoA carboxylase activity. These enzymes are proposed to be the main CO2-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.
2010
The production of valuable biological products from sugars via fermentation is of importance in the chemical industry. One key parameter by which processes are evaluated is the product carbon yield on substrate. In the normal growth of heterotrophic organisms on fermentable sugars, some carbon is lost as CO2. In the production of acetate from hexose or pentose sugars, homoacetogens using the reductive acetyl CoA pathway have a theoretical yield of acetate on glucose of 3. We hypothesized that the addition of enzymes necessary for fixation of the lost CO2 would enhance the theoretical carbon yield of acetate in the dark in organisms without the reductive acetyl CoA pathway. Using computational flux balances of central metabolism of Escherichia coli, we show that the addition of the enzymes for the reductive pentose phosphate (Calvin Benson Bassham) cycle increases the theoretical yield of acetate from glucose or xylose. An even more significant increase in the theoretical carbon yiel...
Carbohydrate Metabolism II: Aerobic Respiration Acetyl-CoA
• Citric Acid Cycle/Krebs Cycle/tricarboxylic acid (TCA) cycle: occurs in mitochondria and functions to oxidize Acetyl-CoA to CO2 and H2O o Cycle produces high-energy molecules: NADH & FADH2 • Acetyl-CoA can be obtained from the metabolism of carbohydrates, fatty acids and amino acids Methods of Forming Acetyl-CoA • Pyruvate dehydrogenase complex: multienzyme compound that catalyzes the reactions which involved pyruvate entering the mitochondrion and subsequently be oxidized and decarboxylated o Three carbon pyruvate is cleaved into a two-carbon acetyl group and a carbon dioxide ▪ Irreversible reaction (glucose cannot be formed directly from Acetyl,CoA o In mammals, complex is made up of five enzymes: ▪ Pyruvate dehydrogenase (PDH) ▪ Dihydrolipoyl transacetylase ▪ Dihydrolipoyl dehydrogenase ▪ Pyruvate dehydrogenase kinase ▪ Pyruvate dehydrogenase phosphatase • Overall reaction is exergonic: negative delta G, and is inhibited by accumulation of Acetyl-CoA and NADH. • Coenzyme A (CoA): written as CoA-SH to show that it is a thiol (with an-SH group) o Acetyl-CoA forms from a covalent attachment between the acetyl group and the-SH group. Results in the formation of thioester o Thioesters are high-energy compounds which are necessary to drive other reactions forward. • Pyruvate Dehydrogenase (PDH): pyruvate is oxidized to yield CO2, and the remaining two-carbon molecules bind covalently to thiamine pyrophosphate (Vitamin B1 or TPP) o TPP is a coenzyme that is non-covalently bonded to TPP o Mg 2+ is also required in this reaction • Dihydrolipoyl Transacetylase: two-carbon molecule that is bound to TPP is oxidized and transferred to lipoic acid Work together to convert pyruvate to Acetyl-CoA Regulate the actions of PDH
PLoS ONE, 2014
Cellular metabolite analyses by 13 C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO 2 aq , mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly 13 C-labelled acetate ( 13 CH 3 -COOH or CH 3 -13 COOH) supported that both the 13 C nuclei give rise to bicarbonate and CO 2 aq . The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO 2 aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO 2 aq , which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO 2 aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.
Proceedings of the National Academy of Sciences, 2008
Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO(2) fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to pyruvate. Pyruvate is converted to phosphoenol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO(2) acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, beta-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-(14)C]4-hydroxybutyrate, [1,4-(13)C(2)], [U-(13)C(4)]succinate, or [1-(13)C]pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO(2) fixation mechanism.