Identification and characterization of Sulfolobus solfataricus D-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner-Doudoroff pathway

Biochem J. 2005 Apr 1;387(Pt 1):271-80. doi: 10.1042/BJ20041053.

Abstract

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner-Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 degrees C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 degrees C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with K(m)=0.45 mM and V(max)=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation-dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner-Doudoroff pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Disaccharides / metabolism
  • Hexoses / metabolism
  • Hydro-Lyases / chemistry*
  • Hydro-Lyases / genetics*
  • Hydro-Lyases / metabolism
  • Molecular Sequence Data
  • Open Reading Frames / genetics
  • Pentoses / metabolism
  • Phosphorylation
  • Sugar Acids / metabolism
  • Sulfolobus solfataricus / enzymology*

Substances

  • Disaccharides
  • Hexoses
  • Pentoses
  • Sugar Acids
  • Hydro-Lyases
  • gluconate dehydratase