Transition from EcoDam to T4Dam DNA Recognition Mechanism without Loss of Activity and Specificity
Transition from EcoDam to T4Dam DNA Recognition Mechanism without Loss of Activity and Specificity
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DOI：
10.1002/cbic.200900441
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发表时间：
2009-10-12
期刊：
影响因子：
3.2
通讯作者：
Jeltsch, Albert
中科院分区：
文献类型：
--
作者：
Elsawy, Hany;Podobinschi, Sveatoslav;Jeltsch, Albert
EcoDam和T4Dam DNA-(腺嘌呤N6)-甲基转移酶都使GATC位点上的腺嘌呤残基甲基化。这些酶在氨基酸序列上高度相关，但它们与目标序列的第一个碱基对的接触有所偏离。EcoDam用K9联系Gual(对应于T4Dam A6)，而T4Dam用R130联系Gual(对应于EcoDam Y138)。我们已将T4Dam DNA识别“移植”到EcoDam中，表明EcoDam K9A/Y138R双突变体具有高度的活性和特异性。我们还研究了这一转变的中间体：EcoDam K9A变体显示出低活性和对Gual的识别丧失[Horton等人，J.Mol。比奥尔。2006、358、559-570]。相比之下，EcoDam Y138R变种携带两个Gual识别元件(来自EcoDam的K9和来自T4Dam的对应于R130的R138)，是完全活跃和特异的。这一结果表明，将EcoDam DNA识别模式转变为T4Dam，而不会失去活性，也不会产生活性降低的进化中间体，这是一条顺利的进化途径。我们一直观察到含有Y138R的EcoDam变体的活性增加；这表明从EcoDam(通过K9的Gual识别)到T4Dam(通过R130的Gual识别)的转变是由选择压力驱动的，以提高催化活性。
The EcoDam and T4Dam DNA-(adenine N6)-methyltransferases both methylate the adenine residue in GATC sites. These enzymes are highly related in amino acid sequence, but they deviate in their contact to the first base pair of the target sequence. EcoDam contacts Gual with K9 (which corresponds to T4Dam A6), while T4Dam contacts Gual with R130 (which corresponds to EcoDam Y138). We have "transplanted" the T4Dam DNA recognition into EcoDam and show that the EcoDam K9A/Y138R double mutant is highly active and specific. We also studied the intermediates of this transition: The EcoDam K9A variant showed low activity and loss of recognition of Gual [Horton, et al., J. Mol. Biol. 2006, 358, 559-570]. In contrast, the EcoDam Y138R variant, which carries both Gual recognition elements (K9 from EcoDam and R138 corresponding to R130 from T4Dam), is fully active and specific. This result indicates that a smooth evolutionary pathway exists for changing the EcoDam DNA recognition mode to T4Dam without loss of activity and without generation of evolutionary intermediates with reduced activity. We consistently observed increased activity of EcoDam variants containing Y138R; this suggests that the transition from EcoDam (Gual recognition through K9) to T4Dam (Gual recognition through R130) was driven by selective pressure towards increased catalytic activity.