Peregrina José R, Herguedas Beatriz, Hermoso Juan A, Martínez-Júlvez Marta, Medina Milagros
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Institute of Biocomputation and Physics of Complex Systems, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Biochemistry. 2009 Apr 14;48(14):3109-19. doi: 10.1021/bi802077c.
Ferredoxin-NADP+ reductases (FNRs) must determine the coenzyme specificity and allow the transient encounter between N5 of its flavin cofactor and C4 of the coenzyme nicotinamide for efficient hydride transfer. Combined site-directed replacements in different putative determinants of the FNR coenzyme specificity were simultaneously produced. The resulting variants were structurally and functionally analyzed for their binding and hydride transfer abilities to the FNR physiological coenzyme NADP+/H, as well as to NAD+/H. The previously studied Y303S mutation is the only one that significantly enhances specificity for NAD+. Combination of mutations from the pyrophosphate or 2'-phosphate regions, even including Y303S, does not improve activity with NAD+, despite structures of these FNRs show how particular coenzyme-binding regions resembled motifs found in NAD+/H-dependent enzymes of the FNR family. Therefore, the "rational approach" did not succeed well, and coenzyme specificity redesign in the FNR family will be more complex than that anticipated in other NADP+/NAD+ families.
铁氧化还原蛋白 - NADP⁺还原酶（FNRs）必须确定辅酶特异性，并允许其黄素辅因子的N5与辅酶烟酰胺的C4之间发生短暂接触，以实现高效的氢化物转移。在FNR辅酶特异性的不同假定决定因素中同时进行了组合定点置换。对所得变体进行了结构和功能分析，以研究它们与FNR生理辅酶NADP⁺/H以及NAD⁺/H的结合和氢化物转移能力。先前研究的Y303S突变是唯一显著增强对NAD⁺特异性的突变。来自焦磷酸或2'-磷酸区域的突变组合，即使包括Y303S，也不能提高对NAD⁺的活性，尽管这些FNR的结构显示了特定的辅酶结合区域如何类似于FNR家族中依赖NAD⁺/H的酶中发现的基序。因此，“理性方法”并未取得很好的效果，FNR家族中的辅酶特异性重新设计将比其他NADP⁺/NAD⁺家族预期的更为复杂。
