Fermani S, Ripamonti A, Sabatino P, Zanotti G, Scagliarini S, Sparla F, Trost P, Pupillo P
Dipartimento di Chimica "G. Ciamician", Università di Bologna, via Selmi 2, Bologna, 40126, Italia.
J Mol Biol. 2001 Nov 30;314(3):527-42. doi: 10.1006/jmbi.2001.5172.
Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis. The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes. Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.
在此，我们报道了与烟酰胺腺嘌呤二核苷酸磷酸（NADP）复合的光合甘油醛-3-磷酸脱氢酶（GAPDH）的首个晶体结构。该酶从菠菜叶绿体中纯化得到，由单一类型的亚基（A）组成同四聚体。它表现出非调节性的依赖NADP和依赖烟酰胺腺嘌呤二核苷酸（NAD）的活性，且更倾向于NADP。通过分子置换法将该结构解析到了3.0埃的分辨率。晶体属于空间群C222，不对称单元中有三个单体。三个单体中的一个利用空间群222点对称性形成一个四聚体，另外两个单体通过非晶体学对称性、利用一个晶体学2重轴形成一个非常相似的四聚体。该蛋白质在辅因子结合结构域和催化结构域的区域与已知的GAPDHs显示出很大的结构同源性。与迄今为止研究的所有其他GAPDHs一样，A(4)-GAPDH属于脱氢酶的罗斯曼折叠家族。然而，与该家族的大多数脱氢酶不同，NADP的腺苷2'-磷酸基团不与周围任何带正电荷的残基形成盐桥，而是通过与属于罗斯曼折叠的一个苏氨酸残基以及位于对称相关单体的S环中的一个丝氨酸残基形成氢键而固定在位。这些结果在增加我们对一种重要光合酶的了解的同时，也有助于对吡啶核苷酸依赖性酶中NADP与NAD识别的总体理解。尽管A(4)-GAPDH的整体结构与细菌和真核生物的胞质GAPDH相似，但叶绿体四聚体很独特，因为它实际上可被视为二聚体的二聚体，因为单体通过跨越半胱氨酸200残基形成的二硫键成对结合。在除菠菜以外的动物、细菌或植物的其他胞质或叶绿体GAPDHs中未发现这种桥。
