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, 193 (5), 1191-200

Labeling and Enzyme Studies of the Central Carbon Metabolism in Metallosphaera Sedula

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Labeling and Enzyme Studies of the Central Carbon Metabolism in Metallosphaera Sedula

Sebastian Estelmann et al. J Bacteriol.

Abstract

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.

Figures

FIG. 1.
FIG. 1.
Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD+); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.
FIG. 2.
FIG. 2.
Incorporation of radioactive label from 4-hydroxy[1-14C]butyrate by autotrophically growing M. sedula cells into metabolites of the carbon fixation cycle and into key metabolites of the central carbon metabolism. Labeled carbon is marked by a box. Only one incomplete turn of the carbon fixation cycle is shown for clarity reasons. The repetitive circulation would result in some scrambling of label, but it would not change the principal ratio of the labeling of pyruvate, oxaloacetate, and 2-oxoglutarate. Note that the second unlabeled acetyl-CoA molecule derived from the tracer 4-hydroxy[1-14C]butyrate is not shown. For the amount of label incorporated, see the text.
FIG. 3.
FIG. 3.
Incorporation of label from [1,4-13C1]succinate into the central metabolites pyruvate, oxaloacetate, and 2-oxoglutarate. The synthesis of 2-oxoglutarate shown here assumes the functioning of (si)-citrate synthase. Labeled carbon atoms are marked by boxes. Compare the labeling pattern to those in Tables 1 and 2.
FIG. 4.
FIG. 4.
Metabolic fluxes in autotrophically grown M. sedula cells. The open arrows symbolize metabolic fluxes leading away from central carbon metabolites to building blocks. The numbers in the arrows indicate the percentages of all biosynthetic fluxes that derive from the central carbon precursors, such as acetyl-CoA, pyruvate, oxaloacetate, etc. The numbers in the ovals refer to the percentage of the individual fluxes of the carbon fixation cycle that has to supply 100% of all biosynthetic central precursor metabolites. Since the cycle has to generate one extra molecule of acetyl-CoA and also regenerates the starting molecule acetyl-CoA, the flux leading to acetyl-CoA sums up to 200% acetyl-CoA formation. The values have to be compared to the total inorganic carbon fixation rate. M. sedula grew autotrophically with a generation time of 15 h, which corresponds to a specific growth rate (μ) of 0.766 h−1, requiring a specific carbon fixation rate of 64 nmol·min−1·mg−1 protein. This estimation is based on the approved equation correlating the specific substrate (S) consumption (dS) per time unit (dt) to the growth rate μ, dS/dt = (μ/YX. Y represents the established growth yield for bacterial cells of 1 g of dry cell mass formed per 0.5 g of carbon fixed. Note that this figure is independent of the growth substrate and depends solely on the fact that ∼50% of bacterial cell dry mass is carbon. X refers to 1 g cell dry mass, and it is a truism that in bacteria ∼50% of cell dry mass is protein; hence, 1 g of cell dry mass corresponds to ∼0.5 g of protein. Assuming that two carbon atoms are fixed in one turn of the cycle, the minimal specific activities of the enzymes of the autotrophic carbon fixation cycle that are required in vivo are about 32 nmol·min−1·mg−1 protein (compare to data in Table 3).

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