Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, 19104.
Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, 19104.
Proc Natl Acad Sci U S A. 2022 Jul 19;119(29):e2119695119. doi: 10.1073/pnas.2119695119. Epub 2022 Jul 12.
Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors β-D-glucose 6-phosphate (G6P) and "catalytic" NADP (NADPc), as well as a "structural" NADP (NADPs) located ∼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADPs in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADPs have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADPc and NADPs in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADPs induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.
人葡萄糖-6-磷酸脱氢酶（G6PD）是细胞内 NADPH 的主要来源，因此在维持还原型谷胱甘肽方面发挥着关键作用，可保护细胞免受溶血性贫血等氧化应激紊乱的影响。G6PD 是一种多聚体酶，使用辅因子β-D-葡萄糖 6-磷酸（G6P）和“催化”NADP（NADPc），以及距活性位点约 25 Å 的“结构”NADP（NADPs），生成 NADPH。尽管 X 射线晶体学和生化研究表明 NADPs 通过稳定多聚体 G6PD 构象在维持催化活性方面发挥作用，但尚未评估 NADPs 的其他潜在作用。在这里，我们确定了在不存在结合配体的情况下人野生型 G6PD 的高分辨率冷冻电子显微镜结构，以及在不存在或存在 G6P 的情况下结合 NADPc 和 NADPs 的催化 G6PD-D200N 突变体的结构。这些结构的比较，以及之前报道的结构，表明未结合配体的人 G6PD 形成具有相似整体折叠的二聚体和四聚体的混合物，并且 NADPs 的结合诱导 C 末端延伸区的结构有序化，并别构调节 G6P 的结合和催化。这些研究对于理解 G6PD 缺乏症以及 G6PD 介导的疾病的治疗具有重要意义。
