Sulfolobus solfataricus P2 is a thermoacidophilic archaeon that metabolizes glucose and galactose via an unusual branched Entner-Doudoroff (ED) pathway, which is characterized by a non-phosphorylative (np-) and a semi-phosphorylative (sp-) branch. However, so far the physiological significance of both pathway branches is unknown. In order to address these question two key enzymes of the branched ED pathway, the class II glycerate kinase (GK) of the np-ED and the 2-keto-3-deoxygluconate kinase (KDGK) of the sp-ED branch in S. solfataricus were investigated. GK was recombinantly purified and characterized with respect to its kinetic properties. Mg2+ dependent Sso-GK (Glycerate + ATP → 2-Phosphoglycerate + ADP) showed unusual regulatory properties, i.e. substrate inhibition and cooperativity by D-glycerate and ATP, and a substrate-inhibition model was established fitting closely to the experimental data. Furthermore, deletion of the sp-ED key enzyme KDGK in S. solfataricus PBL2025 resulted in a similar growth phenotype on glucose as substrate compared to the wild type. In contrast, the mutant showed strongly increased concentrations of np-ED intermediates whereas the hexose and pentose phosphates as well as trehalose were decreased. Together the results indicate that (i) the np-ED pathway is able to compensate for the missing sp-ED branch in glucose catabolism, (ii) that in addition to its catabolic function the sp-ED pathway has an additional although not essential role in providing sugar phosphates for anabolism/gluconeogenesis and (iii) that GK, with its unusual regulatory properties seems to play a major role in controlling the flux between the glycolytic np-ED and glycolytic/gluconeogentic sp-ED pathway.

Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles e.g. in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and α-ketoglutaric semialdehyde dehydrogenase (α-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)+ dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD+, SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)+). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and α-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in γ-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO- SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218 methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD+- and CoA-dependent oxidation of methylmalonate semialdehyde (MMSA), malonate semialdehyde (MSA) as well as propionaldehyde (PA).For MSDH a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from Bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.

Within the SulfoSYS (Sulfolobus Systems Biology) project, the effect of temperature on a metabolic network is investigated at the systems level. Sulfolobus solfataricus utilizes an unusual branched ED (Entner–Doudoroff) pathway for sugar degradation that is promiscuous for glucose and galactose. In the course of metabolic pathway reconstruction, a glucose dehydrogenase isoenzyme (GDH-2, SSO3204) was identified. GDH-2 exhibits high similarity to the previously characterized GDH-1 (SSO3003, 61%amino acid identity), but possesses different enzymatic properties, particularly regarding substrate specificity and catalytic efficiency.
In contrast with GDH-1, which exhibits broad substrate specificity for C5 and C6 sugars, GDH-2 is absolutely specific for glucose. The comparison of kinetic parameters suggests that GDH-2 might represent the major player in glucose catabolism via the branched ED pathway, whereas GDH-1 might have a dominant role in galactose degradation via the same pathway as well as in different sugar-degradation pathways,