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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.

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.

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The overall structure and organization of Gnd1. (A) The cartoon representation of Gnd1 homodimer. The citrate molecules are shown in sticks and colored according to atom types, C is green and O is red. (B) The Gnd1 monomer contains three domains, domain A, B and C colored in green, cyan and red, respectively. The figures were made using PyMOL [34].
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Figure 2: The overall structure and organization of Gnd1. (A) The cartoon representation of Gnd1 homodimer. The citrate molecules are shown in sticks and colored according to atom types, C is green and O is red. (B) The Gnd1 monomer contains three domains, domain A, B and C colored in green, cyan and red, respectively. The figures were made using PyMOL [34].

Mentions: The overall structure of the dimer of S. cerevisiae 6PGDH/Gnd1 enzyme with two molecules of citrate is illustrated in Figure 2A. The structure of Gnd1 (PDB code: 2P4Q) was determined by molecular replacement using sheep 6PGDH as the starting model (PDB code: 1PGP). The structure was determined at 2.37 Å resolution. The final model of each monomer contains residues 1–476, two citrate molecules and 212 water molecules (Table 1).


Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1.

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

The overall structure and organization of Gnd1. (A) The cartoon representation of Gnd1 homodimer. The citrate molecules are shown in sticks and colored according to atom types, C is green and O is red. (B) The Gnd1 monomer contains three domains, domain A, B and C colored in green, cyan and red, respectively. The figures were made using PyMOL [34].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The overall structure and organization of Gnd1. (A) The cartoon representation of Gnd1 homodimer. The citrate molecules are shown in sticks and colored according to atom types, C is green and O is red. (B) The Gnd1 monomer contains three domains, domain A, B and C colored in green, cyan and red, respectively. The figures were made using PyMOL [34].
Mentions: The overall structure of the dimer of S. cerevisiae 6PGDH/Gnd1 enzyme with two molecules of citrate is illustrated in Figure 2A. The structure of Gnd1 (PDB code: 2P4Q) was determined by molecular replacement using sheep 6PGDH as the starting model (PDB code: 1PGP). The structure was determined at 2.37 Å resolution. The final model of each monomer contains residues 1–476, two citrate molecules and 212 water molecules (Table 1).

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