Maier Johannes A H, Albu Razvan F, Jurkowski Tomasz P, Jeltsch Albert
Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany.
Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany.
Biochimie. 2015 Dec;119:60-7. doi: 10.1016/j.biochi.2015.10.011. Epub 2015 Oct 22.
CcrM-related DNA-(adenine N6)-methyltransferases play very important roles in the biology of Caulobacter crescentus and other alpha-proteobacteria. These enzymes methylate GANTC sequences, but the molecular mechanism by which they recognize their target sequence is unknown. We carried out multiple sequence alignments and noticed that CcrM enzymes contain a conserved C-terminal domain (CTD) which is not present in other DNA-(adenine N6)-methyltransferases and we show here that deletion of this part abrogates catalytic activity and DNA binding of CcrM. A mutational study identified 7 conserved residues in the CTD (out of 13 tested), mutation of which led to a strong reduction in catalytic activity. All of these mutants showed altered DNA binding, but no change in AdoMet binding and secondary structure. Some mutants exhibited reduced DNA binding, but others showed an enhanced DNA binding. Moreover, we show that CcrM does not specifically bind to DNA containing GANTC sequences. Taken together, these findings suggest that the specific CcrM-DNA complex undergoes a conformational change, which is endergonic but essential for catalytic activity and this step is blocked by some of the mutations. Moreover, our data indicate that the CTD of CcrM is involved in DNA binding and recognition. This suggests that the CTD functions as target recognition domain of CcrM and, therefore, CcrM can be considered the first example of a δ-type DNA-(adenine N6)-methyltransferase identified so far.
与CcrM相关的DNA（腺嘌呤N6）甲基转移酶在新月柄杆菌和其他α-变形菌的生物学过程中发挥着非常重要的作用。这些酶使GANTC序列甲基化，但其识别靶序列的分子机制尚不清楚。我们进行了多序列比对，发现CcrM酶含有一个保守的C末端结构域（CTD），而其他DNA（腺嘌呤N6）甲基转移酶中不存在该结构域，并且我们在此表明删除该部分会消除CcrM的催化活性和DNA结合能力。一项突变研究在CTD中鉴定出7个保守残基（在13个测试残基中），其中任何一个发生突变都会导致催化活性大幅降低。所有这些突变体都表现出DNA结合能力改变，但AdoMet结合能力和二级结构没有变化。一些突变体表现出DNA结合能力降低，而另一些则表现出DNA结合能力增强。此外，我们表明CcrM不会特异性结合含有GANTC序列的DNA。综上所述，这些发现表明特定的CcrM-DNA复合物会发生构象变化，这是一个吸能过程，但对催化活性至关重要，并且这一步骤会被一些突变所阻断。此外，我们的数据表明CcrM的CTD参与DNA结合和识别。这表明CTD作为CcrM的靶标识别结构域发挥作用，因此，CcrM可被视为迄今为止鉴定出的δ型DNA（腺嘌呤N6）甲基转移酶的首个实例。
