微生物學(xué)通報        MAY 20, 2008, 35(5): 661~665
Microbiology            2008 by Institute of Microbiology, CAS
[email protected]

柳廣飛*  周集體  周  覓  李敬美

(大連理工大學(xué)環(huán)境與生命學(xué)院  大連  116023)

摘  要: 利用同源建模構(gòu)建了Rhodobacter sphaeroides的偶氮還原酶AZR的三級結(jié)構(gòu)模型, AZR為一種   型結(jié)構(gòu)的黃素氧化還原蛋白,。兩類依賴黃素的偶氮還原酶的三級結(jié)構(gòu)對比表明它們具有高度的相似性。在序列和結(jié)構(gòu)對齊分析的基礎(chǔ)上, 選擇保守位點K109進行K109A和K109H的定點突變研究。突變后K109H的最適pH=6, 而K109A的最適pH=9,。突變未改變AZR的最適溫度(30℃)。第109位正電荷殘基對甲基紅的結(jié)合有重要影響; 而K109H對NADPH的結(jié)合并非保守突變,。K109可能只參與對NADPH的2'-磷酸基團的結(jié)合, 而對NADH的結(jié)合無影響,。

關(guān)鍵詞: 偶氮還原酶, 同源建模, 定點突變

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  /  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 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

全文下載：偶氮還原酶AZR的結(jié)構(gòu)及其K109的定點突變研究

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