Catalytic improvement and structural analysis of atrazine chlorohydrolase by site-saturation mutagenesis
Catalytic improvement and structural analysis of atrazine chlorohydrolase by site-saturation mutagenesis
复制标题
位点饱和诱变莠去津氯水解酶的催化改进和结构分析
DOI：
10.1080/09168451.2016.1156481
复制
发表时间：
2016-03
影响因子：
1.6
通讯作者：
Chen Defu
中科院分区：
文献类型：
--
作者：
Guo Yuan;Zhao Panjie;Zhang Wenhao;Li Xiaolong;Chen Xiwen;Chen Defu
为了提高阿特拉津氯水解酶（AtzA）的催化活性，以参与底物结合的氨基酸残基（Gln71）和催化效率（Val12、Ile393和Leu395）为目标，建立了位点饱和诱变文库。基于雨淋红球菌的筛选得到17个变异，其特异活性比野生型高1.2 ~ 5.2倍。在这些变异中，Gln71倾向于被疏水氨基酸取代，Ile393和Leu395分别被极性氨基酸取代，尤其是精氨酸，Val12分别被丙氨酸取代。Q71R和Q71M通过扩大底物进入通道和影响n -乙基结合显著降低Km。位点393和395的突变显著增加了kcat/Km，可能是由于氢键形成，提高了双β-sheet结构域和整个酶的稳定性。此外，还讨论了Gln71突变提高底物亲和力与双β-片结构域修饰提高催化效率之间的矛盾关系。图形摘要：AtzA变体的结构修改。
To improve the catalytic activity of atrazine chlorohydrolase (AtzA), amino acid residues involved in substrate binding (Gln71) and catalytic efficiency (Val12, Ile393, and Leu395) were targeted to generate site-saturation mutagenesis libraries. Seventeen variants were obtained through Haematococcus pluvialis-based screening, and their specific activities were 1.2–5.2-fold higher than that of the wild type. For these variants, Gln71 tended to be substituted by hydrophobic amino acids, Ile393 and Leu395 by polar ones, especially arginine, and Val12 by alanine, respectively. Q71R and Q71M significantly decreased the Km by enlarging the substrate-entry channel and affecting N-ethyl binding. Mutations at sites 393 and 395 significantly increased the kcat/Km, probably by improving the stability of the dual β-sheet domain and the whole enzyme, owing to hydrogen bond formation. In addition, the contradictory relationship between the substrate affinity improvement by Gln71 mutation and the catalytic efficiency improvement by the dual β-sheet domain modification was discussed. Graphical abstract Structrual modification in AtzA variants.
登录
查看更多内容
影响因子：
14.9
作者：
Wass MN;Kelley LA;Sternberg MJ
通讯作者：
Sternberg MJ
影响因子：
4.8
作者：
Cai, Yuanheng;Bhuiya, Mohammad-Wadud;Liu, Chang-Jun
通讯作者：
Liu, Chang-Jun
DOI：
10.1111/febs.13384
发表时间：
2015-10
期刊：
The FEBS Journal
影响因子：
--
作者：
D. Bandyopadhyay;M. Murthy;H. Balaram;P. Balaram
通讯作者：
D. Bandyopadhyay;M. Murthy;H. Balaram;P. Balaram
影响因子：
4.4
作者：
C. Scott;C. Jackson;C. Coppin;R. Mourant;Margaret E. Hilton;T. Sutherland;R. Russell;J. Oakeshott
通讯作者：
C. Scott;C. Jackson;C. Coppin;R. Mourant;Margaret E. Hilton;T. Sutherland;R. Russell;J. Oakeshott
影响因子：
3.6
作者：
Sajid A. Noor;F. Changey;J. Oakeshott;C. Scott;F. Martin-Laurent
通讯作者：
Sajid A. Noor;F. Changey;J. Oakeshott;C. Scott;F. Martin-Laurent