一个enantioselective环氧化酶的定向进化:在每个进化阶段揭示enantioselectivity的来源
Manfred T Reetz1, Marco Bocola, Li-Wen Wang
1Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mulheim/Ruhr, Germany. reetz@mpi-muelheim.mpg.de
Journal of the American Chemical Society
|May 28, 2009
概括
定向进化创造了一种优异的环氧化酶酶突变体,用于溶解种族性甘氨基烯乙烯. 这种增强的酶显示出显著改善的酶选择性,为性催化提供了更绿色的替代方案.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 有机化学 有机化学
- 结构生物学 结构生物学
背景情况:
- 定向进化为传统的不对称催化剂提供了替代方案.
- 酶可以被设计成能产生酶选择性催化剂.
- 环氧化酶是有价值的生物催化剂.
研究的目的:
- 阐明Aspergillus niger环氧化酸酶的定向进化突变体中增强的反抗选择性的机制基础.
- 了解野生类型和突变酶之间的结构和功能差异.
- 为了验证基质范围和工程酶的酶选择性预测.
主要方法:
- 动态分析 动态分析
- 分子动力学模拟的模拟.
- 分子建模分子建模
- 抑制测定试验 抑制测定
- 野生类型和突变酶的X射线晶体学
主要成果:
- 突变的环氧化酶表现出与野生类型 (E=4.6) 相比,对glycidyl phenyl ether分辨率的反选择性 (E=115) 显著增加.
- 尽管整体结构相似,但X射线结构显示了野生类型和突变酶之间活性位点结构的显著差异.
- 计算和实验数据支持一个模型,其中突变的活性部位优先结合并定位 (S) -enantiomer进行水解.
结论:
- 突变酶的增强酶选择性归因于特定活性位点的修改,有利于 (S) -enantiomer的生产性结合.
- 定向进化是优化生物催化剂中的酶酶选择性的强大工具.
- 该研究提供了一种机械的理解,使得能够预测相关基质的酶性能.
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