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.

FIGURE 1.

The non-phosphorylative Entner-Doudoroff pathway for the catabolism of d-glucose and d-galactose in S. solfataricus.

FIGURE 2.

The proposed non-phosphorylative Entner-Doudoroff pathway for the catabolism of d-xylose and l-arabinose in S. solfataricus. The scheme proposed in this paper is shown in solid arrows. The xylose dehydrogenase is the same protein as that labeled glucose dehydrogenase in Fig. 1, the one enzyme being catalytically active with d-glucose, d-galactose, d-xylose, and l-arabinose. The KDG-aldolase is proposed to catalyze the aldol cleavage of KD-gluconate and KD-galactonate (see Fig. 1) and of KD-xylonate and KD-arabinonate. The direct conversion of KD-xylonate to 2-oxoglutarate, as proposed by Brouns et al. (8), is shown with dotted arrows. DOP dehydrogenase, 2,5-dioxopentanoate dehydrogenase.

FIGURE 3.

Enzyme activities in cell extracts of S. solfataricus. Cell extracts were prepared from S. solfataricus grown on d-glucose and on d-xylose. The catalytic activities in these two extracts of the enzymes in Fig. 2 were assayed as described under “Experimental Procedures.” Glycolaldehyde oxidoreductase was assayed with DCPIP as electron acceptor. DOP dehydrogenase is 2,5-dioxopentanoate dehydrogenase and was assayed with pentanedial as substrate.

FIGURE 4.

Anion exchange chromatography of a cell extract from S. solfataricus. A cell extract was prepared from S. solfataricus grown on d-xylose and subjected to anion exchange chromatography on a HiTrap Q-Sepharose column using 50 mm Tris-HCl, pH 8, and an elution gradient of 0–0.7 M NaCl. The elution profile (A_{280 nm}) between 0.13 and 0.35 m NaCl is shown. All fractions were assayed for gluconate dehydratase (gray columns) and xylonate dehydratase (black columns), and the total activity (μmol product per min) in each fraction is displayed. mAU, milliabsorbance units.