Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

Abstract

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...

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

“…Accordingly, all three enzyme activities [GDH, KD(P)A, and glycolaldehyde:OR] were found to be unchanged upon growth on D-glucose and D-xylose, respectively. Conversely, those enzyme activities specific for pentose catabolism, i.e., the conversion of KDX/KDA to ␣-KG and malate, were found to be induced during growth on D-xylose (291). Dehydratase activities, i.e., XAD/AraD and GAD, which appear to be specific for pentose and hexose breakdown, respectively, seem not to be regulated in response to the growth substrate.…”

“…Also, glycolaldehyde is shuttled into the CAC after oxidation to glycoxylate via glycolate and further conversion to malate via malate synthase. In Archaea, pentose utilization has been reported for some aerobic halophiles and for Sulfolobus species (14,283,287,291,343,345). For other Archaea, including hyperthermophiles from the orders Thermococcales, Archaeoglobales, Thermoproteales, Desulfurococcales, and Pyrodictyales, no growth on pentoses has been reported, although many of these organisms are able to grow with hexoses or hexose polymers as carbon and energy sources (14).…”

“…In contrast to SsoGDH-1 also converting C 5 sugars, SsoGAD did not convert C 5 sugar acids like D/L-xylonate or D/L-arabinonate (289). For the breakdown of C 5 sugars, other dehydratases have been identified, i.e., xylonate dehydratase (XAD) (putatively SSO2665) and arbinonate dehydratase (AraD; SSO3124) (see below) (291). Sequence comparison revealed that SsoGAD (GnaD) belongs to the enolase-like superfamily, and it was predicted to contain typical enolase-like N-and C-terminal domains.…”

“…volcanii; the induction of AraDH, AraD, a novel 2-keto-3-deoxyarabinonate dehydratase (KDAD), and an ␣-KGSADH at the transcriptional as well as translational levels has been reported; and the recombinant enzymes have been characterized (283) (Fig. 16B) (291). The enzymes from S. solfataricus, except for AOR and XAD, were recombinantly produced and biochemically characterized.…”

“…The enzymes from S. solfataricus, except for AOR and XAD, were recombinantly produced and biochemically characterized. From these experiments, it was proposed that the same enzymes, i.e., GDH and KD(P)G aldolase (see above), involved in hexose (D-glucose and D-galactose) degradation via the branched ED pathway, are also employed in pentose (D-xylose and L-arabinose) degradation due to their promiscuous properties toward C 6 and C 5 sugars and the corresponding keto sugar acids, respectively (291). Furthermore, the gylcolaldehyde OR activity was argued to represent a side activity of GA:OR from the npED branch.…”