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

Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H2 under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7). 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 ... In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase. Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]). From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO2 acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).