偶氮还原酶AZR的结构及其K的定点突变研究.PDFVIP

微生物学通报 MAY 20, 2008, 35(5): 661~665 Microbiology © 2008 by Institute of Microbiology, CAS tongbao@ 研究报告 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 ( 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。 关键词: 偶氮还原酶, 同源建模, 定点突变 Structure Modeling of Azoreductase AZR and 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 con- structed 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 opti- mal 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 invo