Serre L, Vellieux F M, Medina M, Gomez-Moreno C, Fontecilla-Camps J C, Frey M
IBS/LCCP, Grenoble, France.
J Mol Biol. 1996 Oct 18;263(1):20-39. doi: 10.1006/jmbi.1996.0553.
The crystal structure of the ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 has been determined at 2.6 A resolution by multiple isomorphous replacement and refined using 15.0 A to 1.8 A data, collected at 4 degrees C, to an R-factor of 0.172. The model includes 303 residues, the flavin adenine dinucleotide cofactor (FAD), one sulfate ion located at the putative NADP+ binding site and 328 water molecule sites. The structure of Anabaena FNR, including FAD, a network of intrinsic water molecules and a large hydrophobic cavity in the C-terminal domain, resembles that of the spinach enzyme. The major differences concern the additional short alpha-helix (residues 172 to 177 in Anabaena FNR) and residues Arg 100 and Arg 233 which binds NADP+ instead of Lys 116 and Lys 244 in the spinach enzyme. Crystals of a complex of Anabaena FNR with NADP+ were obtained. The model of the complex has been refined using 15 A to 2.25 A X-ray data, collected at -170 degrees C, to an R-factor of 0.186. This model includes 295 residues, FAD, the full NADP+ (with an occupancy of 0.8) and 444 water molecules. The 2'-5' adenine moiety of NADP+ binds to the protein as 2'-phospho-5'-AMP to the spinach FNR. The nicotinamide moiety is turned towards the surface of the protein instead of stacking onto the FAD isoalloxazine ring as would be required for hydride transfer. The model of the complex agrees with previous biochemical studies as residues Arg 100 and Arg 233 are involved in NADP+ binding and residues Arg77, Lys 53 and Lys 294, located on the FAD side of the enzyme, remain free to interact with ferredoxin and flavodoxin, the physiological partners of ferredoxin: NADP reductase.
通过多同晶置换法，在2.6埃分辨率下测定了蓝藻鱼腥藻PCC 7119的铁氧化还原蛋白：NADP⁺还原酶（FNR）的晶体结构，并使用在4℃收集的15.0埃至1.8埃数据进行精修，最终R因子为0.172。该模型包含303个残基、黄素腺嘌呤二核苷酸辅因子（FAD）、位于假定的NADP⁺结合位点的一个硫酸根离子以及328个水分子位点。鱼腥藻FNR的结构，包括FAD、内部水分子网络和C端结构域中的一个大疏水腔，与菠菜酶的结构相似。主要差异在于额外的短α螺旋（鱼腥藻FNR中的残基172至177）以及结合NADP⁺的精氨酸100和精氨酸233，而不是菠菜酶中的赖氨酸116和赖氨酸244。获得了鱼腥藻FNR与NADP⁺复合物的晶体。使用在-170℃收集的15埃至2.25埃X射线数据对复合物模型进行精修，最终R因子为0.186。该模型包含295个残基、FAD、完整的NADP⁺（占有率为0.8）和444个水分子。NADP⁺的2'-5'腺嘌呤部分以2'-磷酸-5'-AMP的形式与菠菜FNR中的蛋白质结合。烟酰胺部分朝向蛋白质表面，而不是像氢化物转移所需的那样堆积在FAD异咯嗪环上。复合物模型与先前的生化研究一致，因为精氨酸100和精氨酸233参与NADP⁺结合，位于酶FAD一侧的精氨酸77、赖氨酸53和赖氨酸294仍可自由地与铁氧化还原蛋白和黄素氧化还原蛋白相互作用，它们是铁氧化还原蛋白：NADP还原酶的生理伙伴。
