UdgX的突变和结构分析:对活动站点口袋架构及其演变的洞察
Shashanka Aroli1, Eui-Jeon Woo2,3, Balasubramanian Gopal4
1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Nucleic acids research
|June 7, 2023
概括
一种 uracil 产生的酶 UdgX 使用独特的 R 循环结构. 突变显示H109和E52是其催化活性的关键,而Q53稳定了R循环,使其与其他UDG区分开来.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 结构生物学 结构生物学
背景情况:
- UdgX从DNA中切除乌拉,与AP-DNA形成共价键.
- UdgX与UDG-4家族 (F4-UDGs) 的结构相似,但具有独特的灵活R-loop.
- 主要图案,包括图案A (GEQPG) 和图案B (HPS) 在UdgX中保留或分离.
研究的目的:
- 研究特定残留物和结构特征在UdgX活动中的功能作用.
- 阐明UdgX与F4-UDGs分离的机制.
- 了解R循环和动图A/B残留物对UdgX功能的贡献.
主要方法:
- UdgX的局部定向突变发生,包括单个和双重突变.
- 对突变蛋白中的UDG活性进行分析.
- 确定UdgX突变体的晶体结构.
- 生物信息分析以比较UdgX与F4-UDGs.
主要成果:
- 在H109 (H109A,H109S等) 的突变. 恢复了传统的UDG活动,表明它在共价键形成中的作用.
- 突变物E52Q,E52N和E52A表明,E52与H109形成了催化二,增强了核友性.
- Q53A突变证实,A动机中的Q53对于稳定R环形状至关重要.
- 基因B中的R184A突变支持其在基质结合中的作用.
- 结构和突变数据揭示了突变的活性部位的拓变化,与UDG活动相关.
结论:
- UdgX通过特定的氨基酸替代,特别是动图A中的Q53,与F4-UDGs分离.
- UdgX独特的R循环结构由Q53稳定,促进其独特的催化机制.
- 催化二 (E52-H109) 和基质结合残留物 (R184) 对于 UdgX 功能至关重要.
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