Observing an induced-fit mechanism during sequence-specific DNA methylation
Observing an induced-fit mechanism during sequence-specific DNA methylation
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DOI：
10.1074/jbc.m607538200
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发表时间：
2006-12-01
影响因子：
4.8
通讯作者：
Reich, Norbert
中科院分区：
文献类型：
--
作者：
Estabrook, R. August;Reich, Norbert
表征驱动诱导拟合机制的构象变化及其对酶特异性的定量重要性对于充分理解酶的功能至关重要。在这里，我们报道了m.h hai，一种序列特异性的DNA胞嘧啶C-5甲基转移酶，它在结合同源DNA时重组一个柔性环（残基80-100），作为诱导配合机制的一部分。为了直接观察这种类似于26埃的构象重排，并为理解其对特异性的重要性提供基础，我们用色氨酸代替环残基Lys-91和Glu-94。双突变体W41F/K91W和W41F/E94W在动力学和热力学性质上相对稳定。W41F/E94W显示出DNA序列依赖性的荧光变化：当酶结合同源DNA时，平衡态和瞬态荧光发生显著变化，而非特异性DNA不存在。这些实时的、基于溶液的结果提供了直接证据，证明与同源DNA的结合诱导环状重组进入封闭的构象，从而导致活性位点的正确组装。我们建议m.h hai扫描环开放构象中的非特异性DNA，并在同源位点被识别后重新排列成封闭形式。荧光数据排除了环运动先于碱基翻转的机制，我们发现环重排与碱基翻转直接耦合，因为目标鸟苷：胞嘧啶碱基对内单氢键的顺序去除导致相应的环运动变化。
The characterization of conformational changes that drive induced-fit mechanisms and their quantitative importance to enzyme specificity are essential for a full understanding of enzyme function. Here, we report on M. HhaI, a sequence-specific DNA cytosine C-5 methyltransferase that reorganizes a flexible loop (residues 80-100) upon binding cognate DNA as part of an induced-fit mechanism. To directly observe this similar to 26 angstrom conformational rearrangement and provide a basis for understanding its importance to specificity, we replaced loop residues Lys-91 and Glu-94 with tryptophans. The double mutants W41F/K91W and W41F/E94W are relatively unperturbed in kinetic and thermodynamic properties. W41F/E94W shows DNA sequence-dependent changes in fluorescence: significant changes in equilibrium and transient state fluorescence that occur when the enzyme binds cognate DNA are absent with nonspecific DNA. These real-time, solution-based results provide direct evidence that binding to cognate DNA induces loop reorganization into the closed conformer, resulting in the correct assembly of the active site. We propose that M. HhaI scans nonspecific DNA in the loop-open conformer and rearranges to the closed form once the cognate site is recognized. The fluorescence data exclude mechanisms in which loop motion precedes base flipping, and we show loop rearrangements are directly coupled to base flipping, because the sequential removal of single hydrogen bonds within the target guanosine: cytosine base pair results in corresponding changes in loop motion.