Metabolic Pathway Promiscuity in the Archaeon Sulfolobus solfataricus Revealed by Studies on Glucose Dehydrogenase and 2-Keto-3-deoxygluconate Aldolase

Abstract

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.The hyperthermophilic Archaeon Sulfolobus solfataricus grows optimally at 80 -85°C and pH 2-4, utilizing a wide range of carbon and energy sources (1). It has become one of the most comprehensively researched model organisms of archaeal metabolism and bioenergetics (2). Central metabolism in this organism involves a modified Entner-Doudoroff pathway (3), production of acetyl-CoA by pyruvate:ferredoxin oxidoreductase (4), and the citric acid cycle coupled to oxidative phosphorylation (5). The modified Entner-Doudoroff pathway is a nonphosphorylative variant of the classic pathway and proceeds with no net production of ATP (Fig. 1). An analogous pathway has also been detected in the thermoacidophilic Archaea Sulfolobus acidocaldarius (6), Thermoplasma acidophilum (7), and Thermoproteus tenax (8), as well as strains of Aspergillus fungi (9, 10).The first reaction of the non-phosphorylative Entner-Doudoroff pathway involves the NAD(P)-dependent oxidation of glucose to gluconate, catalyzed by glucose dehydrogenase. Gluconate is then dehydrated by gluconate dehydratase to 2-keto-3-deoxygluconate (KDG), 1 which undergoes an aldolate cleavage to pyruvate and glyceraldehyde, catalyzed by KDG aldolase. Glyceraldehyde dehydrogenase then oxidizes glyceraldehyde to glycerate, which is phosphorylated by glycerate kinase to give 2-phosphoglycerate. A second molecule of pyruvate is produced from this by the actions of enolase and pyruvate kinase.Glucose dehydrogenase has previously been purified to homogenei...

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

“…starch, glucose, arabinose, fructose ;Grogan 1989) and relies on the modified branched ED pathway for glucose catabolism (Ahmed et al 2005). In the common shunt of the branched ED pathway, glucose dehydrogenase first oxidizes glucose to glucono-lactone (Giardina et al 1986;Lamble et al 2003), which is subsequently converted to gluconate either via a spontaneous reaction or via an enzymatic conversion by glucono-lactonase. Gluconate dehydratase then catalyzes the dehydration of gluconate to 2-keto-3-deoxygluconate (KDG) (Ahmed et al 2005;Kim and Lee 2005;Lamble et al 2004;Verhees et al 2003).…”

“…Gluconate dehydratase then catalyzes the dehydration of gluconate to 2-keto-3-deoxygluconate (KDG) (Ahmed et al 2005;Kim and Lee 2005;Lamble et al 2004;Verhees et al 2003). In the npED branch, the bifunctional 2-keto-3-deoxy-(6-phospho)-gluconate (KD(P)G) aldolase, which is a key player in both branches, cleaves KDG into pyruvate and glyceraldehyde (Buchanan et al 1999;Lamble et al 2003;Lamble et al 2005;Ahmed et al 2005). Glyceraldehyde is then oxidized to glycerate by glyceraldehyde dehydrogenase Jung and Lee 2006) or glyceraldehyde oxidoreductase (Kardinahl et al 1999;Mukund and Adams 1991;Schicho et al 1993;Selig and Schönheit 1994).…”

“…GAP is further processed via the common lower shunt of the EMP pathway finally forming a second molecule of pyruvate. Interestingly, this branched ED pathway was shown to be promiscuous for glucose and galactose in S. solfataricus (Lamble et al 2003(Lamble et al , 2005Theodossis et al 2004, Milburn et al 2006.…”

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

“…Xylose dehydrogenase activity was determined as previously described (6). Assays were performed at 70°C in 1 ml of 100 mM HEPES, pH 8.0.…”

“…Because recombinantly produced glucose dehydrogenase has good activity with the pentose sugars D-xylose and L-arabinose (6) and KDG-aldolase catalyzes the condensation of the C-2 compound glycolaldehyde and pyruvate to form a 2-keto-3-deoxypentanoate, 5 both enzymes are likely to have a role in the catabolism of the naturally occurring pentose sugars, D-xylose and L-arabinose, as well as of the hexose sugars D-glucose and D-galactose. However, the purified gluconate dehydratase was found to be specific for gluconate and galactonate (7).…”