具有可调节基质结合口袋并存于开放金属协调位点的血红酶的设计
Indrek Kalvet1,2,3, Mary Ortmayer4, Jingming Zhao4
1Institute for Protein Design, University of Washington, 3946 W Stevens Way NE, Seattle, Washington 98195, United States.
Journal of the American Chemical Society
|June 21, 2023
概括
科学家设计了一种新型的血结合蛋白, dnHEM1, 这种蛋白质支架可用于各种催化活动的定制酶设计,包括熟练的过氧化酶和反选择性碳转移酶功能.
科学领域:
- 生物化学
- 蛋白质工程
- 酶催化
背景情况:
- 由于其铁-氨酸辅因子和开放的基质结合口袋,血酶如P450表现出了显著的催化多功能性.
- 之前的新设计的血结合蛋白缺乏类似P450的基质口袋,限制了它们的催化作用范围.
研究的目的:
- 设计一个新的蛋白质支架 (dnHEM1),将P450s的催化位体几何与定制蛋白质设计的灵活性相结合.
- 创建一个高亲和度的血结合蛋白,具有可调节的远端口袋,用于基质相互作用和反应性中间体生成.
主要方法:
- 一个螺旋束脚手架的新型蛋白质设计,以与轴向基因连接剂结合一个血红系因子.
- 在1.6 Å分辨率下测定设计蛋白质 (dnHEM1) 的结构的X射线晶体学.
- 通过修改远端口袋来创建过氧化酶和碳转移酶活动来进行蛋白质工程.
主要成果:
- dnHEM1的晶体结构与设计模型非常一致, 具有关键的编程元素.
- 经过工程改造的dnhem1变体表现出具有稳定的中性费里尔中间体的精通过氧化酶活性.
- 重新设计的 dnHEM1 实现了高度选择性的 styrene 环化 (产量高达 93%, 97:3 e.r.) 通过碳转移酶的活性.
结论:
- 开发的新型蛋白质设计方法可以创建具有定制辅助因子和结合口袋的定制酶.
- 这种策略允许精确的酶功能工程,扩大生物催化和合成化学的可能性.
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