偶氮还原酶A的ZR的结构及其K的定点突变研究.doc

偶氮还原酶AZR的结构及其K109的定点突变研究 柳广飞* 周集体 周 觅 李敬美 (大连理工大学环境与生命学院 大连 116023) 摘 要: 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???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(0℃)。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。Site-directed Mutagenesis of Its K109 Residue LIU Guang-Fei* ZHOU Ji-Ti ZHOU Mi LI Jing-Mei (Institute of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116023) Abstract: Three-dimensional structure model of azoreductase AZR of Rhodobacter sphaeroides was constructed using homology modeling method. It is a ?flavodoxin adopting ?/??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 temperature (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. Keywords: Azoreductase, Homology modeling, Site-directed mutagenesis 偶氮染料是分子中具有一个或多个偶氮基的芳香类化合物, 异咯嗪环Sepharose 4B购自Amersham; 偶氮染料甲基红、酸性红14由大连理工大学染料合成实验室提供。 1.3 同源建模 利用Swiss PdbViewer软件, 选择Project Mode, 以基于SWISS-MODEL服务器的同源建模方法构建偶氮还原酶AZR的三级结构模型。利用PyMol软件完成对模型的相关分析。 1.4 定点突变 以此前构建的质粒pGEX-AZR[6]为模板, 利用大引物法经过3次PCR, 实现K109A和K109H突变。突变所用引物如下(下划线标记引入突变位置), pGEX5’: 5′-GGGCTGGCAAGCCACGTTTGGTG-3′; pGEX3’: 5′-GGGCTGGCAAGCCACGTTTGGTG-3′; K109H F: 5′-TGGTGGCGGTCACGGTGGAATAAA TG-3′; K109H R: 5′-TTCCACCGTGACCGCCACCA GCAAC-3′; K109A F: 5′-TGGTGGCGGTGCAGG TGGAATAAATG-3′; K109A R: 5′-TTCCACCTG