Publications

• Cloning, sequencing and expression of the gene encoding glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum.

  Bright J.R., Byrom D., Danson M.J., Hough D.W., Towner P.

  The gene encoding glucose dehydrogenase has been identified by Southern analysis of doubly restricted genomic Thermoplasma acidophilum DNA, using two redundant 17-residue oligonucleotide probes reverse translated from protein N-terminal sequence data. A 1670-bp BamH1-EcoR1 restriction fragment was ... >> More

  The gene encoding glucose dehydrogenase has been identified by Southern analysis of doubly restricted genomic Thermoplasma acidophilum DNA, using two redundant 17-residue oligonucleotide probes reverse translated from protein N-terminal sequence data. A 1670-bp BamH1-EcoR1 restriction fragment was ligated into pUC19 and pUC18 (constructs pTaGDH1 and pTaGDH2, respectively) and cloned in Escherichia coli. The sequence of the whole fragment was determined, and a 1059-bp open reading frame identified as the gene encoding glucose dehydrogenase. Cell-free extracts from E. coli carrying construct pTaGDH1 displayed glucose dehydrogenase activity indistinguishable from controls, but extracts from cells carrying pTaGDH2 displayed a 600-fold increase in glucose dehydrogenase activity. For high-level expression and purification of native protein, the glucose dehydrogenase coding sequence was subcloned into pMEX8. Glucose dehydrogenase purified from E. coli expressing the pMEX8 construct was indistinguishable by SDS/PAGE, N-terminal amino-acid sequence and kinetic analysis from the native enzyme purified from Tp. acidophilum. The derived 352-amino-acid sequence shows less than 20% identity with the glucose dehydrogenases of Bacillus subtilis and Bacillus megaterium but, by comparison with other eubacterial and eukaryotic dehydrogenase sequences, a portion of its sequence has been tentatively identified as a cofactor-binding region. << Less

  Eur. J. Biochem. 211:549-554(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase.

  Lamble H.J., Heyer N.I., Bull S.D., Hough D.W., Danson M.J.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 278:34066-34072(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

  Smith L.D., Budgen N., Bungard S.J., Danson M.J., Hough D.W.

  Glucose dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme is a tetramer of polypeptide chain Mr 38,000 +/-3000, it is catalytically active with both NAD+ and NADP+ cofactors, and it is thermostable and remarkably resistant to ... >> More

  Glucose dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme is a tetramer of polypeptide chain Mr 38,000 +/-3000, it is catalytically active with both NAD+ and NADP+ cofactors, and it is thermostable and remarkably resistant to a variety of organic solvents. The amino acid composition was determined and compared with those of the glucose dehydrogenases from the archaebacterium Sulfolobus solfataricus and the eubacteria Bacillus subtilis and Bacillus megaterium. The N-terminal amino acid sequence of the Thermoplasma acidophilum enzyme was determined to be: (S/T)-E-Q-K-A-I-V-T-D-A-P-K-G-G-V-K-Y-T-T-I-D-M-P-E. << Less

  Biochem. J. 261:973-977(1989) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Glucose dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

  Giardina P., de Biasi M.G., de Rosa M., Gambacorta A., Buonocore V.

  Glucose dehydrogenase has been purified to homogeneity from cell extracts of the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme utilizes both NAD+ and NADP+ as coenzyme and catalyses the oxidation of several monosaccharides to the corresponding glyconic acid. Substra ... >> More

  Glucose dehydrogenase has been purified to homogeneity from cell extracts of the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme utilizes both NAD+ and NADP+ as coenzyme and catalyses the oxidation of several monosaccharides to the corresponding glyconic acid. Substrate specificity and oxidation rate depend on the coenzyme present; when NAD+ is used, the enzyme binds and oxidizes specifically sugars presenting equatorial orientation of hydroxy groups at C-2, C-3 and C-4. The Mr of the native enzyme is 124,000 and decreases to about 60,000 in the presence of 6 M-guanidinium chloride and to about 30,000 in the presence of 5% (w/v) SDS. The enzyme shows maximal activity at pH 9, 77 degrees C and 20 mM-Mg2+, -Mn2+ or -Ca2+ and is fairly stable in the presence of chaotropic agents and water-miscible organic solvents such as methanol or acetone. << Less

  Biochem. J. 239:517-522(1986) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase.

  Siebers B., Wendisch V.F., Hensel R.

  Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first ... >> More

  Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the 13C label in alanine synthesized by cells grown with [1-13C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD+ and NADP+ as cosubstrates, showing a 100-fold higher affinity for NADP+. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP+. A striking feature was the influence of NADP+ and NAD+ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases. << Less

  Arch. Microbiol. 168:120-127(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: enzyme purification, characterisation and N-terminal sequence.

  Bonete M.J., Pire C., Llorca F.I., Camacho M.L.

  An NAD(P)-glucose dehydrogenase from the extremely halophilic Archaeon, Haloferax mediterranei, has been purified to electrophoretic homogeneity. The purified enzyme has been characterised with respect to its cofactor specificity, subunit composition and its salt and thermal stability. The N-termi ... >> More

  An NAD(P)-glucose dehydrogenase from the extremely halophilic Archaeon, Haloferax mediterranei, has been purified to electrophoretic homogeneity. The purified enzyme has been characterised with respect to its cofactor specificity, subunit composition and its salt and thermal stability. The N-terminal amino acid sequence has been determined and N-terminus alignment with sequences of other glucose dehydrogenases shows that the halophilic enzyme most closely resembles the NAD(P)-linked glucose dehydrogenase from the thermophilic Archaeon Thermoplasma acidophilum. However, the halophilic glucose dehydrogenase appears to be a dimeric protein, in contrast to the tetrameric enzyme from the thermophile. << Less

  FEBS Lett. 383:227-229(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Heterologous overexpression of glucose dehydrogenase from the halophilic archaeon Haloferax mediterranei, an enzyme of the medium chain dehydrogenase/reductase family.

  Pire C., Esclapez J., Ferrer J., Bonete M.J.

  The first gene encoding a glucose dehydrogenase (GDH) from a halophilic organism has been sequenced. Amino acid sequence alignments of GDH from Haloferax mediterranei show a high degree of homology with the thermoacidophilic GDHs and with other enzymes from the medium chain dehydrogenase/reductase ... >> More

  The first gene encoding a glucose dehydrogenase (GDH) from a halophilic organism has been sequenced. Amino acid sequence alignments of GDH from Haloferax mediterranei show a high degree of homology with the thermoacidophilic GDHs and with other enzymes from the medium chain dehydrogenase/reductase family. Heterologous overexpression using the mesophilic organism Escherichia coli as the host has been performed and the expression product was obtained as inclusion bodies. To obtain the halophilic enzyme in its native form refolding and reactivation in a saline environment were required. A pure and highly concentrated sample of the enzyme was obtained using a purification procedure based on the protein's halophilicity. This method may be useful as a general procedure for purifying other halophilic proteins from mesophilic hosts. << Less

  FEMS Microbiol. Lett. 200:221-227(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.