Kinetics of Methylation by EcoP1I DNA Methyltransferase.
Kinetics of Methylation by EcoP1I DNA Methyltransferase.
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DOI：
10.4061/2010/302731
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发表时间：
2010-07-15
期刊：
影响因子：
--
通讯作者：
Desirazu NR
中科院分区：
文献类型：
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作者：
Bheemanaik S;Sistla S;Krishnamurthy V;Arathi S;Desirazu NR
EcoP 1 I DNA MTase（M.EcoP1I）是噬菌体P1的N6-腺嘌呤MTase，属于EcoP 1 I限制-修饰（R-M）系统的一部分，属于III型R-M系统。它识别序列5′-AGACC-3′并甲基化内部腺嘌呤。M.EcoP1I需要Mg 2+将甲基转移到DNA。M.EcoP1I显示在溶液中作为二聚体存在，并且即使在高盐浓度（0.5M）下，二聚体M.EcoP1I也不解离成单体，这表明单体亚基之间的强相互作用。与M.EcoP1I的预孵育和同位素分配研究表明，双链体DNA首先结合，然后是双链体Met的动力学机制。有趣的是，M.EcoP1I甲基化的DNA底物中存在的Mn 2+和Ca 2+而不是Mg 2+与不同的亲和力。氨基酸分析和金属离子存在下的甲基化试验表明，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]。EcoP 1 I DNA MTase使用含有一个以上识别位点的DNA上的甲基化的分布模式催化甲基基团的转移。EcoP 1 I DNA MTase使用N-乙基马来酰亚胺的化学修饰导致酶活性的不可逆失活，这表明半胱氨酸残基在催化中的可能作用。
EcoP1I DNA MTase (M.EcoP1I), an N6-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 Mg2+ 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 Mn2+ and Ca2+ other than Mg2+ 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 [358ID(x)n … ExK401 and 600DxDxD604 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.