偶氮还原酶AZR的结构及其K^(109)的定点突变研究

Structure Modeling of Azoreductase AZR and Site-directed Mutagenesis of Its K^(109) Residue

利用同源建模构建了Rhodobacter sphaeroides的偶氮还原酶AZR的三级结构模型,AZR为一种α/β型结构的黄素氧化还原蛋白。两类依赖黄素的偶氮还原酶的三级结构对比表明它们具有高度的相似性。在序列和结构对齐分析的基础上,选择保守位点K109进行K109A和K109H的定点突变研究。突变后K109H的最适pH=6,而K109A的最适pH=9。突变未改变AZR的最适温度(30℃)。第109位正电荷残基对甲基红的结合有重要影响;而K109H对NADPH的结合并非保守突变。K109可能只参与对NADPH的2’-磷酸基团的结合,而对NADH的结合无影响。

Three-dimensional structure model of azoreductase AZR of Rhodobacter sphaeroides was constructed using homology modeling method. It is a flavodoxin adopting α/β structure. Structure alignment of two different types of flavin-dependent azoreductases revealed that they possessed high similarity. Based on sequence and structure analysis, site-directed mutagenesis of K109H and K109A were performed. The optimal pH values are pH 6 and pH 9 for K109H and K109A mutant protein, respectively. The optimal tempera- ture （30℃） is not affected by mutagenesis. Positively charged residues at position 109 is necessary for the binding of methyl red, while K109H is not a conserved mutagenesis for the binding of NADPH. K109 may only be involved in the binding of the 2＇-phosphate group of NADPH and have no effect on the binding of NADH.