Publications

• The Bacillus subtilis yqjI gene encodes the NADP+-dependent 6-P-gluconate dehydrogenase in the pentose phosphate pathway.

  Zamboni N., Fischer E., Laudert D., Aymerich S., Hohmann H.P., Sauer U.

  Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known ... >> More

  Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme. << Less

  J. Bacteriol. 186:4528-4534(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of glucose-6-phosphate dehydrogenase (NADP+/NAD+) and 6-phosphogluconate dehydrogenase (NADP+/NAD+) from methanol-grown Pseudomonas C.

  Ben-Bassat A., Goldberg I.

  Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADPH+ 1-oxidoreductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase, EC 1.1.1943) have been purified from methanol-grown Pseudomonas C. Glucose-6-phosphate dehydrogenase exhibits activity ... >> More

  Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADPH+ 1-oxidoreductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase, EC 1.1.1943) have been purified from methanol-grown Pseudomonas C. Glucose-6-phosphate dehydrogenase exhibits activity with either NADP+ or NAD+ as coenzymes, V NADP+ = 0.96 V NAD+.Km values of 22, 290, and 250 microns are obtained for NADP+, NAD+ and glucose 6-phosphate (NADP+ as the coenzyme), respectively. ATP inhibits Glc-6P dehydrogenase activity with NAD+ as coenzyme and to a less extent the activity with DANP+. In the presence of MgCl2, ATP inhibition of Blc-6P dehydrogeanse activity is abolished. 6-Phosphogluconate dehydrogenase has a dual specificity for both NADP+ or NAD+ as coenzymes, V NADP+ = 1.66 V NAD+.Km values of 20, 500 and 100 microns are obtained for NADP+, NAD+ and 6-phosphogluconate (NADP+ as the coenzyme), respectively. With NAD+ as the coenzyme ATP inhibits 6-phosphogluconate dehydrogeanse activity, while with NADP+ as the coenzyme, activity was less affected. The possible role of these enzymes in the metabolism of one-carbon (C1)-compounds in Pseudomonas C is discussed and compared with other methylotrophic microorganisms. << Less

  Biochim Biophys Acta 611:1-10(1980) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The gnd gene encoding a novel 6-phosphogluconate dehydrogenase and its adjacent region of Actinobacillus actinomycetemcomitans chromosomal DNA.

  Yoshida Y., Nakano Y., Yamashita Y., Koga T.

  A 10-kb DNA fragment containing the gnd gene from Actinobacillus actinomy-cetemcomitans Y4 was isolated and sequenced. The structural gnd gene codes for 6-phosphogluconate dehydrogenase that consists of 484 amino acids. In contrast to the gnd gene in Escherichia coli, Salmonella typhimurium, or Kl ... >> More

  A 10-kb DNA fragment containing the gnd gene from Actinobacillus actinomy-cetemcomitans Y4 was isolated and sequenced. The structural gnd gene codes for 6-phosphogluconate dehydrogenase that consists of 484 amino acids. In contrast to the gnd gene in Escherichia coli, Salmonella typhimurium, or Klebsiella pneumoniae, the gnd gene of A. actinomycetemcomitans was not located in the rfb or cps operon. The zwf gene encoding glucose 6-phosphate dehydrogenase, which is another enzyme consisting of pentose-phosphate pathway, sided at 3.8-kb upstream from the gnd gene. A phylogenetic tree based on sequence analyses showed higher homology of 6-phospho-gluconate dehydrogenase of A. actinomycetemcomitans with the eucaryotic enzymes rather than with bacterial enzymes. << Less

  Biochem. Biophys. Res. Commun. 230:220-225(1997) [PubMed] [EuropePMC]

• Conformational changes associated with cofactor/substrate binding of 6-phosphogluconate dehydrogenase from Escherichia coli and Klebsiella pneumoniae: Implications for enzyme mechanism.

  Chen Y.Y., Ko T.P., Chen W.H., Lo L.P., Lin C.H., Wang A.H.

  6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the oxidative decarboxylation of 6-phosphogluconate, making ribulose 5-phosphate, along with the reduction of NADP(+) to NADPH and the release of CO(2). Here, we report the first apo-form crystal ... >> More

  6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the oxidative decarboxylation of 6-phosphogluconate, making ribulose 5-phosphate, along with the reduction of NADP(+) to NADPH and the release of CO(2). Here, we report the first apo-form crystal structure of the pathogenic Klebsiella pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10 degrees rotation and resulting in a 7A movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme, which in turn gives rise to concomitant movements of two important NADP(+)-interacting amino acids, M14 and N102. We propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits, involving several related amino acid residues. << Less

  J. Struct. Biol. 169:25-35(2010) [PubMed] [EuropePMC]

• 6-phospho-D-gluconate dehydrogenase from Pseudomonas fluorescens. Properties and subunit structure.

  Stournaras C., Maurer P., Kurz G.

  1. The 6-phospho-D-gluconate dehydrogenase (decarboxylating) (EC 1.1.1.44) from Pseudomonas fluorescens, a B-side stereospecific enzyme, is active with both NAD+ and NADP+, having a specific activity of the homogeneous enzyme of 121 mumols NADH and 23 mumols NADPH, respectively, formed min-1 mg pr ... >> More

  1. The 6-phospho-D-gluconate dehydrogenase (decarboxylating) (EC 1.1.1.44) from Pseudomonas fluorescens, a B-side stereospecific enzyme, is active with both NAD+ and NADP+, having a specific activity of the homogeneous enzyme of 121 mumols NADH and 23 mumols NADPH, respectively, formed min-1 mg protein-1. The pI of the native enzyme is 4.62, the pH optimum is about 8.2. 2. The molecular weight of the native enzyme has been determined to be 126000 by sedimentation equilibrium studies. The molecular weight of the polypeptide chains composing the enzyme has been found to be 32000 by dodecylsulfate/polyacrylamide gel electrophoresis and 31000 by sedimentation equilibrium studies in presence of 6 M guanidine hydrochloride. The native enzyme is composed of four polypeptide chains. 3. Reacting enzyme centrifugation studies gave at pH 8.2 a sedimentation coefficient s20, w of 8.04 S and a diffusion coefficient D20, w of 6.56 F, resulting in a molecular weight of 115000 for the catalytically active form. Thus, the enzyme is active as the tetramer. So far the enzyme from P. fluorescens is the sole 6-phospho-D-gluconate dehydrogenase (decarboxylating) composed of four polypeptide chains. << Less

  Eur J Biochem 130:391-396(1983) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase.

  Yoon H., Anderson C.D., Anderson B.M.

  Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chr ... >> More

  Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of Mr 70,000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2'-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed. << Less

  Biochim Biophys Acta 994:75-80(1989) [PubMed] [EuropePMC]

• Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1.

  He W., Wang Y., Liu W., Zhou C.Z.

  <h4>Background</h4>As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). ... >> More

  <h4>Background</h4>As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.<h4>Results</h4>The crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 A resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-alpha helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 +/-9 microM for 6-phosphogluconate and of 35 +/-6 microM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution.<h4>Conclusion</h4>The overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors. << Less

  BMC Struct. Biol. 7:38-38(2007) [PubMed] [EuropePMC]

• Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+.

  Levy H.R., Vought V.E., Yin X., Adams M.J.

  Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain. NADP+ was modeled into ... >> More

  Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain. NADP+ was modeled into the structure by superimposing the beta alpha beta domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adams et al., 1994, Structure 2, 651-658), enabling us to identify Arg-46 as a potentially important residue for NADP+ binding. Using site-directed mutagenesis, we constructed mutant enzymes in which Arg-46 was replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties. The principal effects in these mutant enzymes were that the Km and Ki values for NADP+ increased by 2 to 3 orders of magnitude over those of the wild-type enzyme. No other kinetic constant was altered more than 6.5-fold. Changing this single amino acid leads to mutant glucose-6-phosphate dehydrogenases with coenzyme specificities that favor NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme. These results confirm that Arg-46 plays a key role in NADP+ binding by contributing a positively charged planar residue that interacts primarily with the 2'-adenosine phosphate. The Arg residue corresponding to Arg-46 in L. mesenteroides glucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes. << Less

  Arch Biochem Biophys 326:145-151(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.