Publications

• NAD+-dependent GAPDH from Thermoproteus tenax - the first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties.

  Brunner N.A., Brinkmann H., Siebers B., Hensel R.

  The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversib ... >> More

  The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway. << Less

  J. Biol. Chem. 273:6149-6156(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of two D-glyceraldehyde-3-phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax.

  Hensel R., Laumann S., Lang J., Heumann H., Lottspeich F.

  Thermoproteus tenax possesses two different glyceraldehyde-3-phosphate dehydrogenases, one specific for NADP+ and the other for NAD+. NADP(H) inhibits the NAD+-specific enzyme competetively with respect to NAD+ whereas NAD(H) virtually does not interact with the NADP+-specific enzyme. Both enzymes ... >> More

  Thermoproteus tenax possesses two different glyceraldehyde-3-phosphate dehydrogenases, one specific for NADP+ and the other for NAD+. NADP(H) inhibits the NAD+-specific enzyme competetively with respect to NAD+ whereas NAD(H) virtually does not interact with the NADP+-specific enzyme. Both enzymes represent homomeric tetramers with subunit molecular masses of 39 kDa (NADP+-specific enzyme) and 49 kDa (NAD+-specific enzyme), respectively. The NADP+-specific enzyme shows significant homology to the known glyceraldehyde-3-phosphate dehydrogenases from eubacteria and eukaryotes as indicated by partial sequencing. The enzymes are thermostable, the NADP+-specific enzyme with a half-life of 35 min at 100 degrees C, the NAD+-specific enzyme with a half-line of greater than or equal to 20 min at 100 degrees C, depending on the protein concentration. Both enzymes show conformational and functional changes at 60-70 degrees C. << Less

  Eur. J. Biochem. 170:325-333(1987) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The semi-phosphorylative Entner-Doudoroff pathway in hyperthermophilic archaea: a re-evaluation.

  Ahmed H., Ettema T.J., Tjaden B., Geerling A.C., van der Oost J., Siebers B.

  Biochemical studies have suggested that, in hyperthermophilic archaea, the metabolic conversion of glucose via the ED (Entner-Doudoroff) pathway generally proceeds via a non-phosphorylative variant. A key enzyme of the non-phosphorylating ED pathway of Sulfolobus solfataricus, KDG (2-keto-3-deoxyg ... >> More

  Biochemical studies have suggested that, in hyperthermophilic archaea, the metabolic conversion of glucose via the ED (Entner-Doudoroff) pathway generally proceeds via a non-phosphorylative variant. A key enzyme of the non-phosphorylating ED pathway of Sulfolobus solfataricus, KDG (2-keto-3-deoxygluconate) aldolase, has been cloned and characterized previously. In the present study, a comparative genomics analysis is described that reveals conserved ED gene clusters in both Thermoproteus tenax and S. solfataricus. The corresponding ED proteins from both archaea have been expressed in Escherichia coli and their specificity has been identified, revealing: (i) a novel type of gluconate dehydratase (gad gene), (ii) a bifunctional 2-keto-3-deoxy-(6-phospho)-gluconate aldolase (kdgA gene), (iii) a 2-keto-3-deoxygluconate kinase (kdgK gene) and, in S. solfataricus, (iv) a GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase; gapN gene). Extensive in vivo and in vitro enzymatic analyses indicate the operation of both the semi-phosphorylative and the non-phosphorylative ED pathway in T. tenax and S. solfataricus. The existence of this branched ED pathway is yet another example of the versatility and flexibility of the central carbohydrate metabolic pathways in the archaeal domain. << Less

  Biochem. J. 390:529-540(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The crystal structure of the allosteric non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeum Thermoproteus tenax.

  Pohl E., Brunner N., Wilmanns M., Hensel R.

  The NAD(+)-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from the hyperthermophilic archaeum Thermoproteus tenax represents an archaeal member of the diverse superfamily of aldehyde dehydrogenases (ALDHs). GAPN catalyzes the irreversible oxidation of d-glyceraldehyd ... >> More

  The NAD(+)-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from the hyperthermophilic archaeum Thermoproteus tenax represents an archaeal member of the diverse superfamily of aldehyde dehydrogenases (ALDHs). GAPN catalyzes the irreversible oxidation of d-glyceraldehyde 3-phosphate to 3-phosphoglycerate. In this study, we present the crystal structure of GAPN in complex with its natural inhibitor NADP(+) determined by multiple anomalous diffraction methods. The structure was refined to a resolution of 2.4 A with an R-factor of 0.21. The overall fold of GAPN is similar to the structures of ALDHs described previously, consisting of three domains: a nucleotide-binding domain, a catalytic domain, and an oligomerization domain. Local differences in the active site are responsible for substrate specificity. The inhibitor NADP(+) binds at an equivalent site to the cosubstrate-binding site of other ALDHs and blocks the enzyme in its inactive state, possibly preventing the transition to the active conformation. Structural comparison between GAPN from the hyperthermophilic T. tenax and homologs of mesophilic organisms establishes several characteristics of thermostabilization. These include protection against heat-induced covalent modifications by reducing and stabilizing labile residues, a decrease in number and volume of empty cavities, an increase in beta-strand content, and a strengthening of subunit contacts by ionic and hydrophobic interactions. << Less

  J. Biol. Chem. 277:19938-19945(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural basis of allosteric regulation and substrate specificity of the non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Thermoproteus tenax.

  Lorentzen E., Hensel R., Knura T., Ahmed H., Pohl E.

  The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic Archaeum Thermoproteus tenax is a member of the superfamily of aldehyde dehydrogenases (ALDH). GAPN catalyses the irreversible oxidation of glyceraldehyde 3-phosphate (GAP) to 3-phosphoglycerate in the ... >> More

  The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic Archaeum Thermoproteus tenax is a member of the superfamily of aldehyde dehydrogenases (ALDH). GAPN catalyses the irreversible oxidation of glyceraldehyde 3-phosphate (GAP) to 3-phosphoglycerate in the modified glycolytic pathway of this organism. In contrast to other members of the ALDH superfamily, GAPN from T.tenax (Tt-GAPN) is regulated by a number of intermediates and metabolites. In the NAD-dependent oxidation of GAP, glucose 1-phosphate, fructose 6-phosphate, AMP and ADP increase the affinity for the cosubstrate, whereas ATP, NADP, NADPH and NADH decrease it leaving, however, the catalytic rate virtually unaltered. As we show here, the enzyme also uses NADP as a cosubstrate, displaying, however, unusual discontinuous saturation kinetics indicating different cosubstrate affinities and/or reactivities of the four active sites of the protein tetramer caused by cooperative effects. Furthermore, in the NADP-dependent reaction the presence of activators decreases the overall S0.5 and increases Vmax by a factor of 3. To explore the structural basis for the different effects of both pyridine nucleotides we solved the crystal structure of Tt-GAPN in complex with NAD at 2.2 A resolution and compared it to the binary Tt-GAPN-NADPH structure. Although both pyridine nucleotides show a similar binding mode, NADPH appears to be more tightly bound to the protein via the 2' phosphate moiety. Moreover, we present four co-crystal structures with the activating molecules glucose 1-phosphate, fructose 6-phosphate, AMP and ADP determined at resolutions ranging from 2.3 A to 2.6 A. These crystal structures reveal a common regulatory site able to accommodate the different activators. A phosphate-binding pocket serves as an anchor point ensuring similar binding geometry. The observed conformational changes upon activator binding are discussed in terms of allosteric regulation. Furthermore, we present a crystal structure of Tt-GAPN in complex with the substrate D-GAP at 2.3 A resolution, which allows us to analyse the structural basis for substrate binding, the mechanism of catalysis as well as the stereoselectivity of the enzymatic reaction. << Less

  J. Mol. Biol. 341:815-828(2004) [PubMed] [EuropePMC]