Hermoso Juan A, Mayoral Tomas, Faro Merche, Gómez-Moreno Carlos, Sanz-Aparicio Julia, Medina Milagros
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química-Física Rocasolano, C.S.I.C., Serrano 119, 28006 Madrid, Spain.
J Mol Biol. 2002 Jun 21;319(5):1133-42. doi: 10.1016/S0022-2836(02)00388-1.
The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyses the production of NADPH in photosynthesis. The three-dimensional structure of FNR presents two distinct domains, one for binding of the FAD prosthetic group and the other for NADP+ binding. In spite of extensive experiments and different crystallographic approaches, many aspects about how the NADP+ substrate binds to FNR and how the hydride ion is transferred from FAD to NADP+ remain unclear. The structure of an FNR:NADP+ complex from Anabaena has been determined by X-ray diffraction analysis of the cocrystallised units to 2.1 A resolution. Structural perturbation of FNR induced by complex formation produces a narrower cavity in which the 2'-phospho-AMP and pyrophosphate portions of the NADP+ are perfectly bound. In addition, the nicotinamide mononucleotide moiety is placed in a new pocket created near the FAD cofactor with the ribose being in a tight conformation. The crystal structure of this FNR:NADP+ complex obtained by cocrystallisation displays NADP+ in an unusual conformation and can be considered as an intermediate state in the process of coenzyme recognition and binding. Structural analysis and comparison with previously reported complexes allow us to postulate a mechanism which would permit efficient hydride transfer to occur. Besides, this structure gives new insights into the postulated formation of the ferredoxin:FNR:NADP+ ternary complex by prediction of new intermolecular interactions, which could only exist after FNR:NADP+ complex formation. Finally, structural comparison with the members of the broad FNR structural family also provides an explanation for the high specificity exhibited by FNR for NADP+/H versus NAD+/H.
黄素酶铁氧化还原蛋白 - NADP⁺还原酶（FNR）在光合作用中催化生成NADPH。FNR的三维结构呈现出两个不同的结构域，一个用于结合FAD辅基，另一个用于结合NADP⁺。尽管进行了广泛的实验并采用了不同的晶体学方法，但关于NADP⁺底物如何与FNR结合以及氢离子如何从FAD转移到NADP⁺等许多方面仍不清楚。通过对共结晶单元进行X射线衍射分析，已确定来自鱼腥藻的FNR:NADP⁺复合物的结构，分辨率达到2.1 Å。复合物形成诱导的FNR结构扰动产生了一个更窄的腔，NADP⁺的2'-磷酸 - AMP和焦磷酸部分在其中完美结合。此外，烟酰胺单核苷酸部分位于FAD辅因子附近形成的一个新口袋中，核糖处于紧密构象。通过共结晶获得的这种FNR:NADP⁺复合物的晶体结构显示NADP⁺处于异常构象，可被视为辅酶识别和结合过程中的中间状态。结构分析以及与先前报道的复合物的比较使我们能够推测出一种允许高效氢化物转移发生的机制。此外，通过预测新的分子间相互作用，该结构为推测的铁氧化还原蛋白:FNR:NADP⁺三元复合物的形成提供了新的见解，这些相互作用仅在FNR:NADP⁺复合物形成后才可能存在。最后，与广泛的FNR结构家族成员的结构比较也为FNR对NADP⁺/H相对于NAD⁺/H表现出的高特异性提供了解释。
