Unusual pathways and enzymes of central carbohydrate metabolism in Archaea

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Sugar-utilizing hyperthermophilic and halophilic Archaea degrade glucose and glucose polymers to acetate or to CO2 using O2, nitrate, sulfur or sulfate as electron acceptors. Comparative analyses of glycolytic pathways in these organisms indicate a variety of differences from the classical Emden–Meyerhof and Entner–Doudoroff pathways that are operative in Bacteria and Eukarya, respectively. The archaeal pathways are characterized by the presence of numerous novel enzymes and enzyme families that catalyze, for example, the phosphorylation of glucose and of fructose 6-phosphate, the isomerization of glucose 6-phosphate, the cleavage of fructose 1,6-bisphosphate, the oxidation of glyceraldehyde 3-phosphate and the conversion of acetyl-CoA to acetate. Recent major advances in deciphering the complexity of archaeal central carbohydrate metabolism were gained by combination of classical biochemical and genomic-based approaches.

Introduction

Comparative biochemical studies on central carbohydrate metabolism revealed that Archaea utilize modifications of the classical Embden–Meyerhof (EM) and Entner–Doudoroff (ED) pathways for glycolysis (Figure 1, Figure 2, Figure 3).

In the classical EM pathway, glucose is converted to fructose-1,6-bisphosphate (FBP), the central intermediate, by way of ATP-dependent hexokinase/glucokinase, phosphoglucose isomerase and ATP-dependent allosteric phosphofructokinases. Cleavage of FBP to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate (GAP) by FBP aldolase and the subsequent isomerization by triosephosphate isomerase yields two mol GAP, which are oxidized to 3-phosphoglycerate by way of phosphorylative glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase; in the latter reaction, ATP is formed by substrate level phosphorylation. 3-Phosphoglycerate is converted to phosphoenolpyruvate (PEP) by way of phosphoglycerate mutase and enolase. The conversion of PEP to pyruvate is catalyzed by allosteric regulated pyruvate kinase, and ATP is formed by substrate level phosphorylation. The net ATP yield of the EM pathway is 2 mol ATP/mol glucose.

In the classical phosphorylative ED pathway, glucose-6-phosphate, which is formed by ATP-dependent glucokinase, is oxidized to 6-phosphogluconate by glucose-6-phosphate dehydrogenase. Subsequent dehydration by 6-phosphogluconate dehydratase yields 2-keto-3-deoxy-(6-phospho)-gluconate (KDPG), the characteristic intermediate of the pathway. KDPG cleavage by KDPG aldolase forms pyruvate and GAP, which is converted to pyruvate by the enzymes also used in the EM pathway. The net ATP yield of ED pathway is 1 mol ATP/mol gucose.

In the hyperthermophilic and the thermophilic aerobic Archaea (Thermoplasma acidophilum [1] and Sulfolobus solfataricus [2, 3, 4•], respectively) glucose is metabolized by way of modifications of the ED pathway, whereas the hyperthermophilic fermentative anaerobes Pyrococcus furiosus, Thermococcus species, Desulfurococcus amylolyticus, the sulfate reducer Archaeoglobus fulgidus strain 7324 and the microaerophilic Pyrobaculum aerophilum use different modifications of the EM pathway ([5, 6, 7, 8]; Reher et al. personal communication). To date, the only Archaeon known to use modifications of both the EM and ED pathways in parallel for glucose degradation is the hyperthermophilic sulfur-dependent anaerobe Thermoproteus tenax [4•, 6, 9, 10, 11, 12•]. In aerobic halophilic Archaea (e.g. Haloarcula marismortui and Halobacterium saccharovorum), 13C-nuclear magnetic resonance (13C-NMR) and enzymatic studies as well as DNA microarray analyses revealed that glucose is degraded only by way of a modified ‘semi-phosphorylative’ ED pathway [13, 14, 15], whereas fructose is almost completely metabolized by way of a modified EM pathway (in Haloarcula marismortui [13] and Haloarcula vallismortis [16]).

Degradation of pyuvate formed by the various glycolytic pathways involves oxidation to acetyl-CoA, which is catalyzed in all Archaea by pyruvate-ferredoxin oxidoreductase. In anaerobic fermentative Archaea, acetyl-CoA is further converted to acetate by an unusual prokaryotic enzyme — ADP-forming acetyl-CoA synthetase (ACD) — whereas in O2-, nitrate- and sulfur-reducing Archaea, acetyl-CoA is oxidized to 2CO2 through the tricarboxylic acid cycle (Figure 1).

Analysis of sugar metabolism in methanogenic Archaea, which are mostly lithoautotrophic H2/CO2- or acetate-utilizing organisms, mainly concerns gluconeogenesis. In some Methanococcus species, the degradation of intracellular glycogen seems to be performed by glycolysis that uses enzymes of a modified EM pathway.

Although most of the catalyzed reactions, and thus the intermediates of the modified archaeal EM and ED pathways, correspond to the classical glycolytic pathways, most of the respective archaeal enzymes show no similarity to their ‘classical’ bacterial and eukaryal counterparts and therefore represent examples of non-homologous gene displacement. Furthermore, the great variety of alternative enzymes, often from different enzyme families, identified in different Archaea (e.g. sugar kinases, phosphoglucose isomerases and enzymes of glyceraldehyde-3-phosphate oxidation) reflects a great metabolic diversity in this third domain of life, which exceeds that of Bacteria and Eukarya.

Recently, great advances were obtained in elucidation of archaeal sugar metabolism for enzymes from (hyper)thermophilic Archaea, because most can be expressed in the mesophilic host Escherichia coli and can subsequently be characterized as recombinant enzymes. Furthermore, the increased intrinsic rigidity of hyperthermophilic proteins obviously favours crystallization and thus structural analysis. However, in halophiles, the adaptation of proteins to high salt concentrations hampers respective developments and therefore less information is available about purified and crystallized enzymes.

In this review, we will present a survey of the archaeal modifications of EM and ED pathways and of the biochemistry and regulation of the unusual enzymes involved. For previous reviews and review-like articles on this topic, see [4•, 6, 12•, 17, 18•, 19, 20, 21, 22].

Section snippets

Glucose phosphorylation

As Archaea lack the bacterial PEP-dependent phophotransferase system-like transport systems, the initial phosphorylation of glucose is the first activation step in the EM modifications. In Archaea, a great variety of enzymes, which differ in specificity and their phosphoryl donor, is observed. In Euryarchaeota, ADP-dependent glucokinases, which exhibit high specificity for glucose, were identified and characterized (e.g. in P. furiosus [5, 23], Thermococcus litoralis [24] and A. fulgidus strain

Modified Entner–Doudoroff pathways in Archaea

Initial studies in Archaea revealed the presence of the non-phosphorylative ED pathway, which involves 2-keto-3-deoxy gluconate (KDG) cleavage, glyceraldehyde oxidation and glycerate phosphorylation in the (hyper)thermophilic Archaea (S. solfataricus [2], T. acidophilum [1] and T. tenax [6, 9, 10, 11]). The semi-phosphorylative ED pathway, which involves KDG kinase, KDPG cleavage and GAP oxidation by GAPDH, was identified as a catabolic route for glucose in halophiles [13, 14]. However, a more

Energetics of modified Embden–Meyerhof and Entner–Doudoroff pathways

The net ATP yields of the classical EM and ED pathway are 2 mol and 1 mol ATP per mol glucose, respectively. The formal net ATP yield of the modified EM pathways that involve non-phosphorylative GAPOR or GAPN is zero (or <1 ATP in T. tenax, assuming that the anabolic-formed PPi, a waste product of the cell, is recycled by PPi-dependent PFK; Figure 2). For Pyrococcus, an additional site of ATP formation has been proposed to occur by way of electron transport phosphorylation coupled to H2 formation

Gluconeogenesis in Archaea

Gluconeogenesis (i.e. glucose 6-phosphate formation from pyruvate) proceeds in lithoautotrophic (e.g. methanogens) and organonotrophic Archaea, as in Bacteria and Eukarya, by way of the reversible reactions of the EM pathway. Accordingly, the irreversible reactions of the modified EM pathway in Archaea (i.e. ADP- and ATP-dependent PFKs, GAPOR and GAPN, and PKs) are reversed by different enzymes: fructose-1,6-bisphosphatase (FBPase), classical GAPDH/phosphoglycerate kinase and

Conclusions

The current analysis of archaeal sugar metabolism and the characterization of the enzymes involved revealed several unusual pathways that are significantly different to the classical EM and ED pathways. In particular, differences in sugar-phosphorylating and -isomerizing enzymes and in GAP-oxidizing enzymes were found. The biochemical, phylogenetic and structural analysis of these novel archaeal enzymes contributes, for example, to the understanding of the complexity of enzyme superfamilies.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

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  • •• of outstanding interest

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