Polyglucose synthesis in Chloroflexus aurantiacus studied by 13C-NMR

Holo, Helge; Grace, D. S. B.

doi:10.1007/bf00456707

Cited by 29 publications

(12 citation statements)

References 14 publications

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“…In line with the experimental data C3 -C5 of ribose directly stem from C1-C3 of the triose phosphatelphosphoenolpyruvate pool by these mechanisms. Notably, the 13C-labeling and coupling patterns of glucose observed by Holo and Grace [14] All experimental findings in the acetate incorporation experiments can be explained satisfactorily if we assume that a significant fraction of malate is formed by the standard glyoxylate cycle and not by the CO, fixation pathway. The key enzymes of the glyoxylate cycle in C. aurantiacus have been demonstrated [15].…”

Section: Labeling Pattern After Feeding Of 3-hydro~y[l-'~c]propionatesupporting

confidence: 58%

Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus

Eisenreich

Strauß

Werz

et al. 1993

European Journal of Biochemistry

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The phototrophic bacterium Chlorojlexus aurantiacus does not use any of the known autotrophic CO, fixation pathways. There is evidence for a new cyclic autotrophic pathway in which acetylCoA is converted to 3-hydroxypropionate and further to succinate and malate. This hypothesis was tested by feeding growing cultures during several generations with 3-hydroxy [ l-13C]propionate, [ l -I3C]acetate, or [2-13C]acetate, in addition to unlabeled CO,. The relative 13C content of individual carbon atoms in biosynthetic amino acids and nucleosides was determined by 'H-and I3C-NMR spectroscopy. I3C coupling patterns were analyzed by two-dimensional 13C-TOCSY experiments which were optimized for the analysis of multiply I3C-labeled biosynthetic samples. From the 13C enrichments of amino acids and nucleosides, the labeling patterns of central metabolic intermediates were evaluated by a retrobiosynthetic approach. Both 3-hydroxypropionate and acetate were incorporated into all central metabolic pools. The I3C labeling and coupling patterns suggest a novel carbon fixation pathway via 3-hydroxypropionate. Specifically, we propose that acetyl-CoA is carboxylated to malonyl-CoA which is reduced under formation of 3-hydroxypropionyl-CoA. Dehydration and reduction yield propionyl-CoA which is converted to succinate by a second carboxylation reaction. The net product of autotrophic carbon fixation appears to be glyoxylate. However, it is not yet known how glyoxylate is channeled into anabolic metabolism. Assimilation of acetate can proceed via the CO, fixation pathway, but also via the glyoxylate pathway.In bacteria, three pathways of autotrophic CO, fixation have been evaluated: the reductive pentose phosphate cycle (Calvin cycle) found in aerobic eubacteria, the reductive citric acid cycle, and the reductive acetyl-CoNcarbon monoxide dehydrogenase pathway found in anaerobic eubacteria and archaebacteria [ 11.Evidence for a fourth autotrophic CO, fixation pathway has recently been presented for Chloroflexus aurantiacus [2, 31, an anaerobic thermophilic phototrophic eubacterium [4, 51. This organism excretes substantial concentrations (5 mM) of 3-hydroxypropionate at the end of autotrophic growth on CO, plus H, [2]; this proves that the bacterium is able to synthesize 3-hydroxypropionate from CO, and H, alone. The question is whether 3-hydroxypropionate is a dead-end product formed from CO, in a side path (e.g. by a peculiar fermentation of storage polyglucose during light limitation in the late growth stage) or whether it is a true intermediate of the novel autotrophic carbon cycle which has not yet been investigated in detail. In the first case, exogenous 3-hydroxy- Traditionally, isotope incorporation studies are interpreted in the forward metabolic direction. In this case, the essential question is whether a given precursor can or can not serve as a precursor for the down-stream product of interest. A different approach was used in the present study. The fed I3C-labeled compounds were incorporated into all amino acids and nu...

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Section: Labeling Pattern After Feeding Of 3-hydro~y[l-'~c]propionatesupporting

confidence: 58%

Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus

Eisenreich

Strauß

Werz

et al. 1993

European Journal of Biochemistry

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“…Materials-Materials were obtained from the commercial sources indicated: [2][3][4][5][6][7][8][9][10][11][12][13][14] C]propionate (1.98 MBq mol Ϫ1 ) from Hartmann Analytic (Braunschweig, Germany), [1,2,[3][4][5][6][7][8][9][10][11][12][13] C 3 ]sodium propionate (99.9% 13 C enrichment) from Cambridge Isotope Laboratories (Andover, MA), acetyl-CoA synthetase, L-lactate dehydrogenase, pyruvate kinase, and myokinase from Roche Diagnostics (Mannheim, Germany). MalonylCoA was prepared as described in Ref.…”

Section: Methodsmentioning

confidence: 99%

“…6B) (2,6). When autotrophically growing cells were fed with [1-13 C 1 ]acetate or [2-13 C 1 ]acetate, the cellular building blocks showed a unique 13 C labeling pattern that could not be explained by any known pathway of carbon metabolism (6).…”

Section: Condensation Of Glyoxylate With Propionylmentioning

confidence: 99%

A Bicyclic Autotrophic CO2 Fixation Pathway in Chloroflexus aurantiacus

Herter¹,

Fuchs²,

Bacher³

et al. 2002

Journal of Biological Chemistry

133

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Phototrophic CO 2 assimilation by the primitive, green eubacterium Chloroflexus aurantiacus has been shown earlier to proceed in a cyclic mode via 3-hydroxypropionate, propionyl-CoA, succinyl-CoA, and malyl-CoA. The metabolic cycle could be closed by cleavage of malylCoA affording glyoxylate (the primary CO 2 fixation product) with regeneration of acetyl-CoA serving as the starter unit of the cycle. Cell extracts of C. aurantiacus were also shown to catalyze the conversion of citramalate into pyruvate and acetyl-CoA in a succinyl-CoAdependent reaction. The data suggest that glyoxylate obtained by the cleavage of malyl-CoA can be utilized by condensation with propionyl-CoA affording erythro-␤-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. This reaction sequence regenerates acetylCoA, which serves as the precursor of propionyl-CoA in the 3-hydroxypropionate cycle. Autotrophic CO 2 fixation proceeds by combination of the 3-hydroxypropionate cycle with the methylmalyl-CoA cycle. The net product of that bicyclic autotrophic CO 2 fixation pathway is pyruvate serving as an universal building block for anabolic reactions.Autotrophic CO 2 fixation in the phototrophic bacterium Chloroflexus aurantiacus has been proposed to proceed via a novel pathway, the 3-hydroxypropionate cycle ( Fig. 1) (1-7). Briefly, acetyl-CoA (1) serves as starting unit, and biotin-dependent carboxylation of acetyl-CoA and propionyl-CoA (4) are the main CO 2 fixation reactions. One turn of the proposed cycle results in conversion of acetyl-CoA into malyl-CoA (8) with consumption of 2 HCO 3 Ϫ and 3 NADPH. Malyl-CoA is cleaved by malyl-CoA lyase with regeneration of the starting molecule acetyl-CoA. Glyoxylate (9) is believed to be the initial CO 2 fixation product (7).The pathway of glyoxylate assimilation into cell material is incompletely understood (5-12). Glycine has been ruled out as an intermediate (7). So far, in vitro transformation of glyoxylate has not been observed, except for pyridine nucleotide-dependent reduction to glycolate (7). An acetyl-CoA-dependent conversion of glyoxylate to malyl-CoA and malate was ascribed to the reverse reaction of malyl-CoA lyase forming malyl-CoA, combined with a side reaction of citrate synthase or acyl-CoA thioesterase, which hydrolyzes malyl-CoA to malate and CoA (7,(13)(14)(15). Previous studies have shown that C. aurantiacus can use pyruvate for anaplerotic reactions (3,7,11,16). Pyruvate is converted to phosphoenolpyruvate (PEP) 1 by pyruvate phosphate dikinase, followed by PEP carboxylation to oxaloacetate by PEP carboxylase. However, pyruvate synthase activity was hardly detectable (12), and the origin of pyruvate in C. aurantiacus is still unknown. To serve as a central intermediate for anaplerotic reactions, it should be formed ultimately from one of the intermediates of the 3-hydroxypropionate cycle and/or from glyoxylate.The aim of this work was to elucidate reactions for glyoxylate assimilation. We show that a reaction sequence starting with glyoxylate and propionyl-CoA afford...

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“…The key enzymes of this cycle were not detectable by other groups, and the 13 C- 12 C isotope discrimination was outside the range of values reported for Calvin cycle organisms (14), indicating the possibility of a different pathway. Indeed, evidence has been accumulated for an alternative carbon fixation cycle, which has been termed the 3-hydroxypropionate cycle (12,14,15,16,39,40). This proposed pathway also seems to operate in the branch of archaebacteria comprising autotrophic Acidianus, Sulfolobus, and Metallosphaera species (4,18,27,30).…”

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confidence: 99%

Autotrophic CO₂Fixation byChloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

et al. 2001

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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.

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Polyglucose synthesis in Chloroflexus aurantiacus studied by 13C-NMR

Cited by 29 publications

References 14 publications

Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus

Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus

A Bicyclic Autotrophic CO2 Fixation Pathway in Chloroflexus aurantiacus

Autotrophic CO₂Fixation byChloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

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Polyglucose synthesis in Chloroflexus aurantiacus studied by 13C-NMR

Cited by 29 publications

References 14 publications

Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus

Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus

A Bicyclic Autotrophic CO2 Fixation Pathway in Chloroflexus aurantiacus

Autotrophic CO2Fixation byChloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

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Autotrophic CO₂Fixation byChloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle