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Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1.

He W, Wang Y, Liu W, Zhou CZ - BMC Struct. Biol. (2007)

Bottom Line: 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.

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Affiliation: Hefei National Laboratory for Physical Sciences at Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, People's Republic of China. dolphinw@mail.ustc.edu.cn <dolphinw@mail.ustc.edu.cn>

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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The binding mode of the two citrate molecules. (A) Electron density of the two citrate molecules FLC1 and FLC2 (2Fo-Fc map contoured at 1.2 σ). (B) A closer look of the conserved residues binding to the two citrate molecules. The C terminal tail of chain B is colored in grey, and chain A in cyan. (C) Superimposed structures of Gnd1 (in cyan) with sheep liver 6PGDH (PDB code: 1PGP; colored in grey). The two citrate molecules (shown in sticks) are superimposed on one molecule of 6PG (shown in sticks) of 1PGP. (D) The surface comparison between yeast Gnd1 bound to two citrate molecules (a, a' and a") and sheep liver 6PGDH monomer bound to 6PG (b, b' and b"). The monomer omitting the bound ligand, the ligand and the complex are shown in a/b, a'/b' and a"/b", respectively.
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Figure 3: The binding mode of the two citrate molecules. (A) Electron density of the two citrate molecules FLC1 and FLC2 (2Fo-Fc map contoured at 1.2 σ). (B) A closer look of the conserved residues binding to the two citrate molecules. The C terminal tail of chain B is colored in grey, and chain A in cyan. (C) Superimposed structures of Gnd1 (in cyan) with sheep liver 6PGDH (PDB code: 1PGP; colored in grey). The two citrate molecules (shown in sticks) are superimposed on one molecule of 6PG (shown in sticks) of 1PGP. (D) The surface comparison between yeast Gnd1 bound to two citrate molecules (a, a' and a") and sheep liver 6PGDH monomer bound to 6PG (b, b' and b"). The monomer omitting the bound ligand, the ligand and the complex are shown in a/b, a'/b' and a"/b", respectively.

Mentions: All the calculations of rotation function and translation function were conducted using the program MOLREP[32] in CCP4 (Correlation coefficient: 53.5%). Refinement was carried out using the programs O and crystallography and NMR system (CNS)[33]. Through the refinement we identified two unexpected electron clouds in the catalytic pocket as citrate molecules used in crystallization. It appeared that two citrate molecules were bound to the enzyme in each monomer (Figure 2A[34], for more details see Figure 3A).


Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1.

He W, Wang Y, Liu W, Zhou CZ - BMC Struct. Biol. (2007)

The binding mode of the two citrate molecules. (A) Electron density of the two citrate molecules FLC1 and FLC2 (2Fo-Fc map contoured at 1.2 σ). (B) A closer look of the conserved residues binding to the two citrate molecules. The C terminal tail of chain B is colored in grey, and chain A in cyan. (C) Superimposed structures of Gnd1 (in cyan) with sheep liver 6PGDH (PDB code: 1PGP; colored in grey). The two citrate molecules (shown in sticks) are superimposed on one molecule of 6PG (shown in sticks) of 1PGP. (D) The surface comparison between yeast Gnd1 bound to two citrate molecules (a, a' and a") and sheep liver 6PGDH monomer bound to 6PG (b, b' and b"). The monomer omitting the bound ligand, the ligand and the complex are shown in a/b, a'/b' and a"/b", respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919378&req=5

Figure 3: The binding mode of the two citrate molecules. (A) Electron density of the two citrate molecules FLC1 and FLC2 (2Fo-Fc map contoured at 1.2 σ). (B) A closer look of the conserved residues binding to the two citrate molecules. The C terminal tail of chain B is colored in grey, and chain A in cyan. (C) Superimposed structures of Gnd1 (in cyan) with sheep liver 6PGDH (PDB code: 1PGP; colored in grey). The two citrate molecules (shown in sticks) are superimposed on one molecule of 6PG (shown in sticks) of 1PGP. (D) The surface comparison between yeast Gnd1 bound to two citrate molecules (a, a' and a") and sheep liver 6PGDH monomer bound to 6PG (b, b' and b"). The monomer omitting the bound ligand, the ligand and the complex are shown in a/b, a'/b' and a"/b", respectively.
Mentions: All the calculations of rotation function and translation function were conducted using the program MOLREP[32] in CCP4 (Correlation coefficient: 53.5%). Refinement was carried out using the programs O and crystallography and NMR system (CNS)[33]. Through the refinement we identified two unexpected electron clouds in the catalytic pocket as citrate molecules used in crystallization. It appeared that two citrate molecules were bound to the enzyme in each monomer (Figure 2A[34], for more details see Figure 3A).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, People's Republic of China. dolphinw@mail.ustc.edu.cn <dolphinw@mail.ustc.edu.cn>

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus