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

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[1-14 C]butyrate and [1,4-13 C 1 ]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 C 4 intermediates 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.

Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO 2 fixation

“…A similar situation occurs in M. sedula, where, by an additional half turn of the cycle, acetyl-CoA plus another two bicarbonate molecules are converted into succinyl-CoA. SuccinylCoA then is converted to malate or oxaloacetate, which can be decarboxylated to pyruvate or phosphoenolpyruvate (27). Hence the precursor metabolites, primarily pyruvate, phosphoenolpyruvate, oxaloacetate, 2-oxoglutarate, and a few others, are generated from succinyl-CoA (Fig.…”

“…C 3 plants lose about 20% of fixed carbon in photorespiration (15,37). † The costs of the synthesis of pyruvate, phosphoenolpyruvate, oxaloacetate, and 2-oxoglutarate in M. sedula vary depending on the pathway of succinyl-CoA conversion to succinate (27).…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…furiosus (198,315) and in obligate and facultative autotrophs such as Ign. hospitalis and Metallosphaera sedula, respectively (316,317). An unusual multimeric form of PEPS has been identified in Staphylothermus marinus but was not functionally characterized in detail (318,319).…”

“…sedula, and a detailed characterization has been reported for the Tco. kodakarensis enzyme (317,330). PCKs belong to the PEP carboxykinase-like superfamily, comprising two major classes, i.e., the ATP-dependent PCKs (ATP-PCKs) and the GTP-dependent PCKs.…”

Archaeal Aldehyde Dehydrogenase ST0064 fromSulfolobus tokodaii, a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase, Is a Succinate Semialdehyde Dehydrogenase

“…SSA exists as an intermediate of the 3HP/4HB cycle and is reduced to 4-hydroxybutylate for further carbon fixation. 17,18) SSADH catalyzes the reaction by which SSA diverges from this pathway and enters the TCA cycle as succinate. An excess amount of SSA, produced by reduction from succinyl-CoA, the only common intermediate of both the 3HP/4HB and the TCA cycle, can be catalytically converted by SSADH to provide carbon sources for central metabolism (Supplemental Fig.…”