We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes D-glucose and D-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.Sulfolobus solfataricus and Sulfolobus acidocaldarius are hyperthermophilic archaea that grow optimally at 78 -85°C, pH 2-4, and are able to utilize a variety of carbon sources, including the four most-commonly occurring sugars in nature, D-glucose, D-galactose, D-xylose, and L-arabinose (1).Metabolism of glucose in S. solfataricus and S. acidocaldarius proceeds via a non-phosphorylative variant of the Entner-Doudoroff pathway, which generates pyruvate with no net production of ATP (Fig. 1) (2-5). Glucose dehydrogenase catalyzes the conversion of glucose to gluconate, which is then dehydrated to 2-keto-3-deoxygluconate (KD-gluconate) 4 by gluconate dehydratase. KD-gluconate in turn is cleaved to pyruvate and glyceraldehyde via 2-keto-3-deoxygluconate aldolase (KDG-aldolase), the glyceraldehyde generating a second molecule of pyruvate via the actions of glyceraldehyde oxidoreductase, glycerate kinase, enolase, and pyruvate kinase.In vitro kinetic analyses of glucose dehydrogenase, gluconate dehydratase, and KDG-aldolase from S. solfataricus showed these enzymes are also capable of catalyzing the catabolism of galactose, the C4 epimer of glucose, to pyruvate and glyceraldehyde, leading to the suggestion that the pathway exhibits a metabolic promiscuity toward these two hexose sugars (5-7). Because recombinantly produced glucose dehydrogenase has good activity with the pentose sugars D-xylose and L-arabinose (6) and KDG-aldolase catalyze...

The pathway of D-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P.

The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by 13C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments. H. saccharolyticus grown on complex media containing glucose or fructose specifically 13C-labeled at C1 and C3, formed acetate and small amounts of lactate. The 13C-labeling patterns, analyzed by 1H- and 13C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea. Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed. After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased. Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose. Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses. The data indicate that, in the archaeon H. saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. Various halophilic archaea, including Haloarcula marismortui and Haloferax volcanii, have been reported to utilize fructose as carbon and energy sources (29,34,46). The pathway of fructose degradation has been studied so far mainly in the Haloarcula species H. vallismortis and H. marismortui (6,7,29). On the basis of enzyme analyses, a modified version of the Embden-Meyerhof (EM) pathway has been proposed, involving fructose phosphorylation via ketohexokinase to fructose-1-phosphate, which is further phosphorylated to fructose-1,6-bisphosphate (FBP) by fructose-1-phosphate kinase (1-PFK). FBP is subsequently cleaved by FBP aldolase (FBA) to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, which are degraded to pyruvate following classical enzymes of the EM pathway. In vivo evidence for the operation of an EM pathway in fructose degradation was demonstrated in H. marismortui by labeling experiments with [13 C]fructose using growing cultures (29). With the same labeling techniques, glucose degradation in H. marismortui was shown to be degraded in vivo via an Entner-Doudoroff (ED) type pathway (29), which is in accordance with the proposed semiphosphorylated ED pathway for glucose degradation in haloarchaea (43).Although several enzymes of the proposed modified EM pathway...

The pathway of L-arabinose degradation was studied in the haloarchaeon Haloferax volcanii. It is shown that L-arabinose is oxidatively degraded to α-ketoglutarate. During growth on L-arabinose, L-arabinose dehydrogenase (L-AraDH) was induced. The enzyme was purified as a 130 kDa homotetrameric protein catalyzing the oxidation of L-arabinose with both NADP(+) and NAD(+). The gene encoding L-AraDH was identified as HVO_B0032 and recombinant L-AraDH showed similar properties as the native enzyme. The L-AraDH deletion mutant did not grow on L-arabinose, but grew unaffected on glucose and D-xylose, indicating a specific involvement in L-arabinose degradation. Phylogenetic analyses attribute the first archaeal L-AraDH to the extended short-chain dehydrogenase/reductase (SDRe) family, where it is part of a novel cluster and thus differs from known archaeal and bacterial pentose dehydrogenases. Further, cell extracts of H. volcanii catalyzed the NADP(+)-dependent conversion of L-arabinoate to α-ketoglutarate. The genes involved in that conversion were identified by analyses of transcripts and deletion mutants as HVO_B0038A, HVO_B0027 and HVO_B0039 recently reported to be involved in D-xylonate conversion to α-ketoglutarate in H. volcanii (Johnsen et al. 2009).