Bheemanaik Shivakumara, Sistla Srivani, Krishnamurthy Vinita, Arathi Sampath, Desirazu Narasimha Rao
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Enzyme Res. 2010 Jul 15;2010:302731. doi: 10.4061/2010/302731.
EcoP1I DNA MTase (M.EcoP1I), an N(6)-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg(2+) for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn(2+) and Ca(2+) other than Mg(2+) with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [(358)ID(x)(n) … ExK(401) and (600)DxDxD(604) motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.
EcoP1I DNA甲基转移酶（M.EcoP1I）是一种来自噬菌体P1的N6 -腺嘌呤甲基转移酶，是EcoP1I限制修饰（R - M）系统的一部分，该系统属于III型R - M系统。它识别5'-AGACC-3'序列并使内部腺嘌呤甲基化。M.EcoP1I将甲基转移到DNA需要Mg(2+)。研究表明，M.EcoP1I在溶液中以二聚体形式存在，即使在高盐浓度（0.5 M）下，二聚体M.EcoP1I也不会解离成单体，这表明单体亚基之间存在强烈的相互作用。对M.EcoP1I进行预孵育和同位素分配研究表明其动力学机制为先结合双链DNA，然后结合S-腺苷甲硫氨酸（AdoMet）。有趣的是，M.EcoP1I在存在Mg(2+)以外的Mn(2+)和Ca(2+)时，也能以不同亲和力使DNA底物甲基化。金属离子存在下的氨基酸分析和甲基化测定表明，M.EcoP1I确实有两个金属离子结合位点[（358）ID(x)(n)…ExK(401)和（600）DxDxD(604)基序]。EcoP1I DNA甲基转移酶在含有多个识别位点的DNA上采用分布甲基化模式催化甲基转移。用N -乙基马来酰亚胺对EcoP1I DNA甲基转移酶进行化学修饰导致酶活性不可逆失活，这表明半胱氨酸残基在催化中可能发挥作用。
