Distal structural elements coordinate a conserved base flipping network.
Distal structural elements coordinate a conserved base flipping network.
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DOI：
10.1021/bi301284f
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发表时间：
2013-02
期刊：
影响因子：
2.9
通讯作者：
Douglas M. Matje;C. Krivacic;F. Dahlquist;N. Reich
中科院分区：
文献类型：
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作者：
Douglas M. Matje;C. Krivacic;F. Dahlquist;N. Reich
在DNA修饰和修复酶中观察到高能量中间体的酶促作用的最引人注目的例证之一，其中单个碱基围绕脱氧核糖磷酸骨架旋转（翻转）180°并进入活性位点。虽然最终状态已被广泛表征，但实验技术尚未对碱基翻转过程和酶所扮演的角色进行完整描述。C5胞嘧啶甲基转移酶M.HhaI协调相互DNA和酶重排的整体，以有效地从DNA螺旋翻转靶胞嘧啶。我们试图了解单个氨基酸在碱基翻转过程中的作用。我们的研究结果表明，M.HhaI在催化环关闭之前启动碱基翻转，并利用催化环中保守的丝氨酸85来加速翻转并维持DNA骨架的扭曲。丝氨酸87在碱基翻转后在DNA螺旋内形成特异性接触，不参与翻转过程或维持催化活性复合物。在催化环的底部，甘氨酸98充当铰链以允许环的构象动力学，并且突变为丙氨酸抑制闭环的稳定。我们的研究结果说明了酶如何利用大量的，远端残基在演唱会上转化为催化底物识别。
One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180° around the deoxyribose-phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The C5 cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.