晶体结构和实验室进化阿尔德海德减少酶的运动研究解释了其新基质特异性的戏剧性转变
Shruthi Sridhar1, Alberto Zavarise1, Tiila Riikka Kiema2
1Department of Chemistry - BMC, Uppsala University, SE-751 23 Uppsala, Sweden.
定向进化创造了一个高度活跃的大肠杆菌FucO酶变体 (DA1472) 用于重的基板. 结构和功能研究解释了它对乙的活性增加了9000倍,增强了生物催化潜力.
科学领域:
- 生物化学 生化学
- 酶学 是一种酶学.
- 蛋白质工程是指蛋白质工程.
背景情况:
- 大肠杆菌酶FucO (Fe2+依赖) 通过使用NADH/NAD+可逆地相互转化 (S) - 乳酸和 (S) - 1,2 - 二醇.
- 野生类型的FucO表现出较差的催化活性与甲等重基质.
研究的目的:
- 为了研究结构和功能基础增强的催化活性在一个有针对性的进化FucO变体 (DA1472) 与重的基板.
- 了解在DA1472突变体中观察到的显著活动增加背后的机制.
主要方法:
- 实验室指导的进化使用乙作为基质.
- 确定DA1472及其母体D93 (L259V) 的晶体结构.
- 用各种基质对野生类型,突变变种和DA1472的催化活性进行系统定量.
主要成果:
- 与野生型FucO相比,N151G/L259V双重突变 (DA1472) 显示甲的kcat/KM增加了9000倍.
- 晶体结构显示了DA1472中活性位点空间的增加,而没有发生主链形状变化.
- 对DA1472与基质模拟物复合的结构分析阐明了大型群体的结合模式.
结论:
- 在DA1472中的氨基酸替代物为重的基质创造了一个更适应性的活性部位,解释了显著的催化增强.
- 结构功能数据提供了关于FucO的基质特异性和金属辅因子替代 (Fe2+与Zn2+) 的局限性的见解.
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