ABSTRACT
The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 353 K and can metabolize glucose and its C4 epimer galactose via a non-phosphorylative variant of the Entner-Doudoroff pathway involving catalytically promiscuous enzymes that can operate with both sugars. The initial oxidation step is catalysed by glucose dehydrogenase (SsGDH), which can utilize both NAD and NADP as cofactors. The enzyme operates with glucose and galactose at similar catalytic efficiency, while its substrate profile also includes a range of other five- and six-carbon sugars. Crystals of the 164 kDa SsGDH homotetramer have been grown under a variety of conditions. The best crystals to date diffract to 1.8 A on a synchrotron source, have orthorhombic symmetry and belong to space group P2(1)2(1)2. Attempts are being made to solve the structure by MAD and MR.
Subject(s)
Glucose 1-Dehydrogenase/chemistry , Sulfolobus solfataricus/enzymology , Archaeal Proteins/chemistry , Archaeal Proteins/isolation & purification , Archaeal Proteins/metabolism , Cloning, Molecular , Crystallization , Escherichia coli/enzymology , Glucose 1-Dehydrogenase/isolation & purification , Glucose 1-Dehydrogenase/metabolism , Hot Temperature , Models, Molecular , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Synchrotrons , Thermodynamics , X-Ray DiffractionABSTRACT
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.
