The phototrophic bacterium ChlorojZexus aurantiucus can grow autotrophically but seems not to assimilate CO, via any of the known autotrophic pathways. Holo [Holo, H. (1989) Arch. Microbiol. 151, 252-2561 proposed a new pathway in which 3-hydroxypropionate is formed from acetylCoA. Previous studies excluded the operation of known CO, fixation pathways and provided indirect evidence for the suggested pathway based on "C-labelling experiments. Here all enzyme activities of the postulated cyclic CO, fixation mechanism are demonstrated in vitro. In essence, acetyl-CoA is carboxylated and reductively converted via 3-hydroxypropionate to propionyl-CoA. PropionylCoA is carboxylated and converted via succinyl-CoA and CoA transfer to malyl-CoA. Malyl-CoA is cleaved to acetyl-CoA and glyoxylate. Thereby, the first CO, acceptor molecule acetyl-CoA is regenerated, completing the cycle and the net CO, fixation product glyoxylate is released. This cycle represents the fourth autotrophic pathway in nature and is designated the 3-hydroxypropionate cycle.Three CO, fixation pathways have been found in autotrophic microorganisms so far (reviewed by [l-71): (a) the reductive pentosephosphate cycle (Calvin cycle) ; (b) the reductive citric acid cycle; (c) and the reductive non-cyclic acetylCoA pathway. Pathway (a) is the only autotrophic pathway in plants and occurs mainly in aerobic bacteria. Pathways (b) and (c) occur in anaerobic bacteria or in those microaerophilic bacteria whose metabolism exhibits anaerobic traits. These pathways require carboxylating and other enzymes which are characteristic and are considered the key enzymes. Key enzymes are ribulose-I ,5-bisphosphate carboxylase and phosphoribulokinase for (a), 2-oxoglutarate synthase and ATP-citrate lyase [8] for (b), and carbon monoxide dehydrogenasehcetyl-CoA synthase for (c).
The unresolvcd autotrophic COz fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubdcterium Chlorojlexus uurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,succinate, and the 3C pattern in cell constituents was determined by 'H-and 13C-NMR spectroscopy of purified amino acids and other cell constituents.In both organisms succinate contributed to < l o % of cell carbon, the major part of carbon originated from C 0 2 . All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic C 0 2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate.The '"C-labelling pattcrn in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic C 0 2 fixation pathway. Surprisingly, the proffercd [l ,4-'3Cl]succinate was partially converted to double-labelled [3,4-"C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under thc growth conditions used, and a reversible citrate cleavage reaction is proposed.The '3C-labelling pattern in C. uuruntiacus disagrees with any of the established C 0 2 fixation pathways; it therefore demands a novel autotrophic COz 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 C02-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.In bacteria three autotrophic C 0 2 fixation pathways are recognized : the reductive pentose phosphate cycle [l], the reductive citric acid cycle [2 -131, and the non-cyclic reductive acetyl-CoA/carbon monoxide dehydrogenase pathway [ 14 -161. The reductive pentose phosphate cycle (Calvin cycle) has been found only in aerobic Eubacteria (for a possible exception see [17]); ribulose-I ,5-bisphosphate carboxylase has not been detected in anaerobes. The two alternative pathways
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...
The biosynthesis of verrucosan-2-ol in the green phototrophic eubacterium Chloroflexus aurantiacus was investigated by in vivo incorporation of singly or doubly 13 C-labeled acetate. The 13 C labeling of the isolated diterpene was analyzed by one-and two-dimensional NMR spectroscopy. The 13 C-labeling patterns of verrucosan-2-ol were compared with the labeling patterns of intermediary metabolites (acetyl-CoA, pyruvate, and glyceraldehyde 3-phosphate) which were deduced from amino acids and nucleosides by retrobiosynthetic analysis. The results show that verrucosan-2-ol is synthesized via mevalonate and not via the deoxyxylulose pathway, which was discovered recently in some eubacteria, algae, and plants. A scheme for the formation of the unusual tetracyclic ring system is offered. The cyclization process is initiated by the solvolysis of pyrophosphate from geranyllinaloyl pyrophosphate and the mechanism involves a Wagner-Meerwein rearrangement, a 1,5-hydride shift, and a cyclopropylcarbinyl to cyclopropylcarbinyl rearrangement.
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