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Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1.
BMC Struct Biol. 2007 Jun 14; 7:38.BS

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.

Authors+Show Affiliations

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>No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17570834

Citation

He, Weiwei, et al. "Crystal Structure of Saccharomyces Cerevisiae 6-phosphogluconate Dehydrogenase Gnd1." BMC Structural Biology, vol. 7, 2007, p. 38.
He W, Wang Y, Liu W, et al. Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1. BMC Struct Biol. 2007;7:38.
He, W., Wang, Y., Liu, W., & Zhou, C. Z. (2007). Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1. BMC Structural Biology, 7, 38.
He W, et al. Crystal Structure of Saccharomyces Cerevisiae 6-phosphogluconate Dehydrogenase Gnd1. BMC Struct Biol. 2007 Jun 14;7:38. PubMed PMID: 17570834.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1. AU - He,Weiwei, AU - Wang,Yi, AU - Liu,Wei, AU - Zhou,Cong-Zhao, Y1 - 2007/06/14/ PY - 2007/03/23/received PY - 2007/06/14/accepted PY - 2007/6/16/pubmed PY - 2007/8/24/medline PY - 2007/6/16/entrez SP - 38 EP - 38 JF - BMC structural biology JO - BMC Struct. Biol. VL - 7 N2 - 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. SN - 1472-6807 UR - https://www.unboundmedicine.com/medline/citation/17570834/Crystal_structure_of_Saccharomyces_cerevisiae_6_phosphogluconate_dehydrogenase_Gnd1_ L2 - https://bmcstructbiol.biomedcentral.com/articles/10.1186/1472-6807-7-38 DB - PRIME DP - Unbound Medicine ER -