Identification and characterization of Sulfolobus solfataricus D-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner-Doudoroff pathway.
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.
Kim
Seonghun
S
School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, South Korea.
Lee
Sun Bok
SB
D000595:Amino Acid Sequence; D004187:Disaccharides / Q000378:metabolism; D006601:Hexoses / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010429:Pentoses / Q000378:metabolism; D010766:Phosphorylation; D013400:Sugar Acids / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology*
D004187:Disaccharides; D006601:Hexoses; D010429:Pentoses; D013400:Sugar Acids; D006836:Hydro-Lyases; C020576:gluconate dehydratase