如何区分N和O:控制甲基转移酶的化学选择性
Emely Jockmann1, Helena Girame2, Wieland Steinchen3,4
1Institute of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany.
概括
被称为S-Adenosyl-l-methionine (SAM) -依赖甲基转移酶 (MTs) 的酶通过不同的构造状态来实现特定的化学选择性. 工程设计这些酶改变了它们的甲基转移偏好,揭示了动态控制的选择性.
科学领域:
- 生物化学 生化学
- 酶学 是一种酶学.
- 结构生物学 结构生物学
背景情况:
- 依赖S-Adenosyl-l-methionine (SAM) 的甲基转移酶 (MTs) 是催化生物通路中甲基转移的关键酶.
- 尽管具有保留的催化机制,但MTs对各种基质和核友来说表现出特定的化学选择性.
- 咖啡酸O-MT (PpCaOMT) 和炭酸N-MT (RgANMT) 具有很高的序列相似性,但在基质和核细胞特异性方面有所不同.
研究的目的:
- 研究酶构造状态在确定甲基转移酶的化学选择性中的作用.
- 为了设计PpCaOMT和RgANMT变体,改变核爱好者的偏好.
- 阐明酶动态,突变和甲基转移选择性之间的关系.
主要方法:
- 针对PpCaOMT和RgANMT的七种变体的合理设计和工程.
- 酶活性测定用于评估甲基转移酶的化学选择性.
- 分子动力学 (MD) 模拟和/交换质谱学 (HDX-MS) 用于分析形态动力学.
主要成果:
- 在PpCaOMT (O-甲基化,封闭状态) 和RgANMT (N-甲基化,开放状态) 中,不同的化学选择性是由不同的构造状态控制的.
- 工程变种表现出改变的核友偏好,其中一些显示出选择性的完全逆转.
- 一种O选择性的ANMT变体显示出明显高于野生型PpCaOMT的催化活性.
- MD模拟和HDX-MS证实,突变调节着形状动态,并影响化学选择性.
结论:
- 酶的结构动态,而不仅仅是活性部位残留物,协同控制甲基转移选择性.
- 调节开放/关闭形状转换是调整MT化学选择性的关键机制.
- 这项研究提供了对具有量身定制基质特异性的酶的合理设计的见解.
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