Characterization of Sulfolobus solfataricus 2-keto-3-deoxy-D-gluconate kinase in the modified Entner-Doudoroff pathway

Abstract

The thermoacidophilic archaeon Sulfolobus solfataricus is known to utilize D-glucose via the nonphosphorylated Entner-Doudoroff (ED) pathway. But, the genome database shows that this microorganism has a gene (kdgK) encoding 2-keto-3-deoxy-D-gluconate (KDG) kinase (KDGK) which phosphorylates KDG to 2-keto-3-deoxy-6-phosphogluconate. Interestingly, kdgK and three other genes in the modified ED pathway are organized as an operon-like structure. In this study, we report confirmation of the catalytic activity of the S. solfataricus KDGK protein. We also found that the kdgK gene was transcribed as polycistronic transcripts. Proteome analysis of cell lysate revealed that all gene products in the kdgK operon were expressed as functional proteins. These results strongly indicate that S. solfataricus metabolizes D-glucose via the 'partially' nonphosphorylated ED pathway. A purified recombinant S. solfataricus KDGK had Km and kcat values of 0.14 mM and 60.8 s-1 respectively for KDG, and showed maximal activity at temperatures between 70 and 80°C and pHs between 7.0 and 8.0.