...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biological chemistry >Functional analysis of amino acid residues at the dimerisation interface of Kpnl DNA methyltransferase
【24h】

Functional analysis of amino acid residues at the dimerisation interface of Kpnl DNA methyltransferase

机译：Kpnl DNA甲基转移酶二聚界面上氨基酸残基的功能分析

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

KpnI DNA-(N-6-adenine) methyltransferase (M.Kpnl) recognises the sequence 5'-GGTACC-3' and transfers the methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.Kpnl exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.Kpnl was obtained based on protein fold-recognition analysis, using the crystal structures of M.Rsrl and M.MbollA as templates. Residues l146, l161 and Y167, the side chains of which are present in the putative dimerisation interface-in the model, were targeted for site-directed mutagenesis. Methylation and in vitro restriction assays showed that the mutant MTases are catalytically inactive. Mutation at the l146 position resulted in complete disruption of the dimer. The replacement of l146 led to drastically reduced DNA and cofactor binding. Substitution of l161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.Kpnl to catalyse the methyl transfer reaction.
机译：KpnI DNA-（N-6-腺嘌呤）甲基转移酶（M.Kpnl）识别序列5'-GGTACC-3'并将甲基从S-腺苷-L-蛋氨酸（AdoMet）转移到腺嘌呤残基的N6位置在每个链中。早期研究表明，M.Kpnl在溶液中以二聚体形式存在，这与大多数其他MTase不同。为了解决二聚化对酶功能的重要性，基于蛋白质折叠识别分析，使用M.Rsrl和M.MbollA的晶体结构作为模板，获得了M.Kpnl的三维模型。在模型中假定的二聚化界面中存在侧链的残基1146、1161和Y167被定点诱变。甲基化和体外限制性酶切试验表明，突变的MTases无催化活性。在l146位的突变导致二聚体的完全破坏。 1146的替换导致DNA和辅因子结合的急剧减少。 1161的取代导致单体之间的相互作用减弱，导致单体和二聚体物种。稳态荧光测量表明，野生型KpnI MTase会诱导结合的DNA发生结构变形，而突变型MTase则不会。结果证实，单体MTase是催化惰性的，二聚化是M.Kpnl催化甲基转移反应的重要事件。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号