Enzyme-promoted base flipping controls DNA methylation fidelity.
Enzyme-promoted base flipping controls DNA methylation fidelity.
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DOI：
10.1021/bi3012912
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发表时间：
2013-03
期刊：
影响因子：
2.9
通讯作者：
Douglas M. Matje;Hongjun Zhou;Darren A Smith;Robert K Neely;D. Dryden;Anita C. Jones;F. Dahlquist;Norbert O. Reich
中科院分区：
文献类型：
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作者：
Douglas M. Matje;Hongjun Zhou;Darren A Smith;Robert K Neely;D. Dryden;Anita C. Jones;F. Dahlquist;Norbert O. Reich
定量了解构象转变如何有助于酶催化和特异性仍然是一个根本性的挑战。一套生物物理方法被用来揭示模型DNA胞嘧啶甲基转移酶M. HhaI的酶-底物复合物的几个瞬态。多维，横向弛豫优化的核磁共振（NMR）实验表明，M.HhaI与非同源和同源DNA序列具有相同的构象。高亲和力同源物样模式需要形成蛋白质-DNA相互作用的子集，其驱动靶碱基从螺旋翻转到活性位点。缺乏这些相互作用的非同源底物经历缓慢的碱基翻转，并且催化环的荧光跟踪证实了在碱基翻转和随后的催化环闭合之前松散的非特异性结合模式的NMR证据。这种缓慢的翻转转变定义了非同源序列甲基化的限速步骤。此外，我们提出了光谱证据的中间沿着基地翻转途径，已被预测，但以前从未观察到。这些发现提供了重要的细节，如何构象重排用于平衡特异性与催化效率。
A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.