Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106-9510, United States.
Biochemistry. 2011 Mar 8;50(9):1465-73. doi: 10.1021/bi101446g. Epub 2011 Feb 10.
The DNA methyltransferase M.HhaI is an excellent model for understanding how recognition of a nucleic acid substrate is translated into site-specific modification. In this study, we utilize direct, real-time monitoring of the catalytic loop position via engineered tryptophan fluorescence reporters to dissect the conformational transitions that occur in both enzyme and DNA substrate prior to methylation of the target cytosine. Using nucleobase analogues in place of the target and orphan bases, the kinetics of the base flipping and catalytic loop closure rates were determined, revealing that base flipping precedes loop closure as the rate-determining step prior to methyl transfer. To determine the mechanism by which individual specific hydrogen bond contacts at the enzyme-DNA interface mediate these conformational transitions, nucleobase analogues lacking hydrogen bonding groups were incorporated into the recognition sequence to disrupt the major groove recognition elements. The consequences of binding, loop closure, and catalysis were determined for four contacts, revealing large differences in the contribution of individual hydrogen bonds to DNA recognition and conformational transitions on the path to catalysis. Our results describe how M.HhaI utilizes direct readout contacts to accelerate extrication of the target base that offer new insights into the evolutionary history of this important class of enzymes.
DNA 甲基转移酶 M.HhaI 是理解如何将核酸底物的识别转化为特异性修饰的优秀模型。在这项研究中，我们利用工程化色氨酸荧光报告器对催化环位置进行直接、实时监测，以剖析在靶胞嘧啶甲基化之前发生在酶和 DNA 底物中的构象转变。我们使用核苷类似物代替靶标和孤儿碱基，确定了碱基翻转和催化环闭合速率的动力学，揭示了碱基翻转先于环闭合，是甲基转移之前的限速步骤。为了确定酶-DNA 界面上单个特定氢键接触如何介导这些构象转变，我们将缺乏氢键基团的核苷类似物掺入识别序列中，以破坏主要沟识别元件。我们确定了四个接触点的结合、环闭合和催化的情况，揭示了单个氢键对 DNA 识别和催化过程中构象转变的贡献存在很大差异。我们的结果描述了 M.HhaI 如何利用直接读出接触来加速靶碱基的脱离，这为理解这一类重要酶的进化历史提供了新的见解。
