He Weiwei, Wang Yi, Liu Wei, Zhou Cong-Zhao
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.
BMC Struct Biol. 2007 Jun 14;7:38. doi: 10.1186/1472-6807-7-38.
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.
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.
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.
作为戊糖磷酸途径的第三种酶，6-磷酸葡萄糖酸脱氢酶（6PGDH）是细胞内NADPH的主要生成者。硫氧还蛋白还原酶和谷胱甘肽还原酶都需要NADPH作为电子供体来还原氧化型硫氧还蛋白或谷胱甘肽（GSSG）。由于硫氧还蛋白和谷胱甘肽是重要的抗氧化剂，因此6PGDH在保护细胞免受氧化应激方面发挥关键作用也就不足为奇了。此外，6PGDH的活性与包括癌症和阿尔茨海默病在内的多种人类疾病有关。三维结构研究对于设计靶向该酶用于潜在治疗应用的小分子将非常有价值。
通过分子置换法，以2.37 Å的分辨率测定了酿酒酵母6-磷酸葡萄糖酸脱氢酶（6PGDH/Gnd1）的晶体结构。Gnd1的整体结构是一个同型二聚体，每个单体有三个结构域，一个具有Rossmann折叠的NADP+结合结构域，一个主要由α螺旋组成的结构域，它对两个亚基之间的疏水相互作用起主要作用，还有一个小的C末端结构域穿透另一个亚基。此外，两个柠檬酸分子占据了每个单体的6PG结合口袋。完整的Gnd1在pH 7.5时，对6-磷酸葡萄糖酸的Km值为50±9 μM，对NADP+的Km值为35±6 μM。但是，缺失Gnd1 C末端35、39或53个残基的截短突变体完全丧失了它们的6PGDH活性，尽管在溶液中仍保留同型二聚体结构。
Gnd1的整体三级结构与其他物种的6PGDH相似。无论是从一级序列比对还是从三维结构叠加来看，底物和辅酶结合位点都高度保守。酶活性测定表明催化作用是按顺序进行的机制，这与先前的研究一致。Gnd1的C末端结构域起到钩子的作用，进一步拉紧二聚体，但它对于二聚化不是必需的。该结构域还充当底物结合口袋的盖子，以控制底物的结合和产物的释放，因此它对于6PGDH活性是不可或缺的。此外，共结晶的柠檬酸分子模拟了底物6-磷酸葡萄糖酸的结合模式，为我们设计6PDGH抑制剂提供了一种新策略。
