Bist Pradeep, Rao Desirazu N
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
J Biol Chem. 2003 Oct 24;278(43):41837-48. doi: 10.1074/jbc.M307053200. Epub 2003 Aug 12.
EcoP15I DNA methyltransferase catalyzes the transfer of the methyl group of S-adenosyl-l-methionine to the N6 position of the second adenine within the double-stranded DNA sequence 5'-CAGCAG-3'. To achieve catalysis, the enzyme requires a magnesium ion. Binding of magnesium to the enzyme induces significant conformational changes as monitored by circular dichroism spectroscopy. EcoP15I DNA methyltransferase was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 8.0. The inactivated enzyme was cleaved into two fragments with molecular masses of 36 and 35 kDa. Using this affinity cleavage assay, we have located the magnesium binding-like motif to amino acids 355-377 of EcoP15I DNA methyltransferase. Sequence homology comparisons between EcoP15I DNA methyltransferase and other restriction endonucleases allowed us to identify a PD(X)n(D/E)XK-like sequence as the putative magnesium ion binding site. Point mutations generated in this region were analyzed for their role in methyltransferase activity, metal coordination, and substrate binding. Although the mutant methyltransferases bind DNA and S-adenosyl-l-methionine as well as the wild-type enzyme does, they are inactive primarily because of their inability to flip the target base. Collectively, these data are consistent with the fact that acidic amino acid residues of the region 355-377 in EcoP15I DNA methyltransferase are important for the critical positioning of magnesium ions for catalysis. This is the first example of metal-dependent function of a DNA methyltransferase. These findings provide impetus for exploring the role(s) of metal ions in the structure and function of DNA methyltransferases.
EcoP15I DNA甲基转移酶催化S-腺苷-L-甲硫氨酸的甲基基团转移至双链DNA序列5'-CAGCAG-3'中第二个腺嘌呤的N6位。为实现催化作用，该酶需要一个镁离子。通过圆二色光谱监测发现，镁与酶的结合会引起显著的构象变化。在pH 8.0、存在抗坏血酸的情况下，微摩尔浓度的硫酸亚铁可使EcoP15I DNA甲基转移酶迅速失活。失活的酶被切割成两个分子量分别为36 kDa和35 kDa的片段。利用这种亲和切割分析方法，我们已将镁结合样基序定位到EcoP15I DNA甲基转移酶的355 - 377位氨基酸上。通过对EcoP15I DNA甲基转移酶与其他限制性内切酶进行序列同源性比较，我们确定了一个PD(X)n(D/E)XK样序列为假定的镁离子结合位点。分析了该区域产生的点突变在甲基转移酶活性、金属配位和底物结合方面的作用。尽管突变型甲基转移酶与DNA和S-腺苷-L-甲硫氨酸结合的能力与野生型酶相同，但它们无活性主要是因为无法翻转靶碱基。总体而言，这些数据与以下事实一致：EcoP15I DNA甲基转移酶中355 - 377区域的酸性氨基酸残基对于催化所需镁离子的关键定位很重要。这是DNA甲基转移酶金属依赖性功能的首个实例。这些发现为探索金属离子在DNA甲基转移酶的结构和功能中的作用提供了动力。
