Youngblood Ben, Shieh Fa-Kuen, De Los Rios Stephanie, Perona John J, Reich Norbert O
Program in Biomolecular Science and Engineering, University of California, Santa Barbara, CA 93106-9510, USA.
J Mol Biol. 2006 Sep 15;362(2):334-46. doi: 10.1016/j.jmb.2006.07.031. Epub 2006 Jul 21.
Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.
通过破坏DNA胞嘧啶甲基转移酶HhaI活性位点与高度保守的柔性环之间疏水界面的相互作用，实现了该酶序列特异性的提高。瞬态荧光实验表明，破坏该界面的突变会使活性位点内螺旋外“翻转”的胞嘧啶碱基定位不稳定。与同源DNA和辅因子类似物S-腺苷-L-高半胱氨酸结合的F124A M.HhaI的三元晶体结构显示，柔性环与酶核心之间的腔体积增加。该腔破坏了环与活性位点之间的界面，从而使螺旋外靶碱基不稳定。碱基翻转的酶-DNA复合物更倾向于回到碱基堆积中间体，导致突变酶比野生型酶对非同源位点的区分能力更强。基于动力学校对的概念以及我们对M.HhaI的理解，我们描述了如何通过甲基转移前中间体的不稳定而不是破坏M.HhaI酶与底物之间的直接相互作用，在环/活性位点界面处通过双突变实现16倍的特异性增强。
