Insights into the autotrophic CO2 fixation pathway of the archaeon Ignicoccus hospitalis: comprehensive analysis of the central carbon metabolism

Abstract

Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo (13)C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO(2) fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO(2) acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the alpha-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.

FIG. 1.

Biosynthetic capacity of I. hospitalis, according to genome data. Circles, putative genes of enzymes that were detected (E value < e−11); squares, genes of enzymes that were not detected in the I. hospitalis genome. The numbers of the putative genes of the I. hospitalis genome are shown: 1a, citrate synthase; 1b, ATP citrate lyase; 2, aconitase; 3, isocitrate dehydrogenase; 4a, malate dehydrogenase; 4b, malate quinone oxidoreductase; 5, fumarate hydratase; 6a, fumarate reductase; 6b, succinate dehydrogenase; 7, succinate-CoA ligase (ADP forming); 8a, 2-oxoacid-ferredoxin oxidoreductase; 8b, acetyl-CoA carboxylase; 8c, pyruvate dehydrogenase; 9a, pyruvate-water dikinase; 9b, pyruvate-phosphate dikinase; 9c, pyruvate kinase; 10a, PEP carboxylase; 10b, PEP carboxykinase; 10c, PEP carboxytransphosphorylase; 11, pyruvate carboxylase; 12, enolase; 13, phosphoglycerate mutase; 14, phosphoglycerate kinase; 15, glyceraldehyde 3-phosphate dehydrogenase; 16, triosephosphate isomerase; 17, fructose 1,6-bisphosphate aldolase; 18, fructose 1,6-bisphosphate phosphatase; 19, phosphoglucose isomerase (cupin type); 20, 3-hexulose 6-phosphate isomerase; 21, 3-hexulose 6-phosphate synthase.

FIG. 2.

Incorporation of ¹⁴C-labeled substrates by growing I. hospitalis cells. (A) [1-¹⁴C]acetate. (B) [1,4-¹⁴C]succinate. (C) [3-¹⁴C]pyruvate. (D) [U-¹⁴C]glucose. The total radioactivities in the assay (▪), in the supernatant/cell-free medium (⧫), and in the cells (▴) in relation to the dry cell mass (×) of the growing I. hospitalis culture are shown.

FIG. 3.

Proposed incomplete pathway of CO₂ fixation in I. hospitalis. The proposed pathway was deduced from the labeling pattern and the complement of enzymes and genes of I. hospitalis. The circles represent highly ¹³C-enriched carbon atoms from [1-¹³C]acetate. Partial randomization of labeling in oxaloacetate is explained by reversible reactions between oxaloacetate, malate, fumarate, and succinate. A reductive carboxylation of succinyl-CoA implies a lack of ¹³C enrichment at C-1 of 2-oxoglutarate. This is at odds with the labeling data for glutamate, proline, and arginine, which all display high ¹³C enrichment at C-1. The compounds in boxes were analyzed.

FIG. 4.

Biosynthesis of histidine via fructose 6-phosphate from two molecules of PEP. (Left column) Formation of ribulose 5-phosphate by oxidative decarboxylation. (Right column) Formation of ribulose 5-phosphate by the ribulose monophosphate pathway. PHI, 3-hexulose 6-phosphate isomerase; HPS, 3-hexulose 6-phosphate synthase. The circles represent ¹³C-enriched carbon atoms from [1-¹³C]acetate. The observed labeling pattern in histidine is shown in the box. The lowercase characters in histidine and ribose phosphate indicate biosynthetically equivalent carbon atoms.

FIG. 5.

Biosynthesis of phenylalanine via fructose 6-phosphate. (Left column) Synthesis of erythrose 4-phosphate by reaction of the nonoxidative pentose phosphate pathway. TK, transketolase; TA, transaldolase. The labeling pattern of dehydroshikimate is constructed from erythrose 4-phosphate and PEP on the basis of the conventional shikimate pathway. (Right column) Synthesis via 6-deoxy-5-ketofructose 1-phosphate and aspartate semialdehyde. The labeling pattern of aspartate semialdehyde was predicted from that of aspartate.

FIG. 6.

Biosynthesis of lysine. (Left column) Formation of lysine via the common pathway starting from aspartate and pyruvate and leading to diaminopimelate. (Right column) Formation of lysine via the uncommon pathway starting from 2-oxoglutarate and acetyl-CoA and leading to α-aminoadipate. The circles represent ¹³C-enriched carbon atoms from [1-¹³C]acetate. The observed labeling pattern in lysine is shown in the box. The labeling pattern of 2-oxoglutarate is reconstructed from the observed pattern in glutamate, proline, and arginine.

FIG. 7.

Biosynthesis of isoleucine. (Left column) Formation of isoleucine via the conventional pathway in which 2-oxobutyrate is formed from threonine. The labeling pattern of threonine corresponds to that of aspartate from which it is formed (Table 3). (Right column) Formation of isoleucine via the unconventional pathway in which 2-oxobutyrate is formed from pyruvate and acetyl-CoA via citramalate and 3-methyloxaloacetate, which is decarboxylated to 2-oxobutyrate. Note that the labeling of 2-oxobutyrate differs in the two pathways. The subsequent steps from 2-oxobutyrate to isoleucine are identical. The circles represent ¹³C-enriched carbon atoms from [1-¹³C]acetate. The observed labeling patterns in threonine and isoleucine are shown in boxes. The labeling pattern of pyruvate is reconstructed from the observed pattern in alanine.