Stopped-flow and mutational analysis of base flipping by the Escherichia coli dam DNA-(adenine-N6)-methyltransferase
Stopped-flow and mutational analysis of base flipping by the Escherichia coli dam DNA-(adenine-N6)-methyltransferase
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DOI：
10.1016/j.jmb.2004.05.033
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发表时间：
2004-08-06
影响因子：
5.6
通讯作者：
Jeltsch, A
中科院分区：
文献类型：
--
作者：
Liebert, K;Hermann, A;Jeltsch, A
通过使用2-氨基嘌呤作为探针检测碱基翻转的停流动力学，我们在这里显示大肠杆菌Dam DNA-（腺嘌呤-N6）-甲基转移酶（MTase）的碱基翻转是一个两相过程：靶碱基翻转非常快（k（flip）> 240 s（-1）），但翻转的碱基结合到酶的活性位点口袋中是缓慢的（k = 0.1-2 s（-1））。而碱基翻转发生在S-腺苷-L-甲硫氨酸（蛋氨酸）的情况下，在活性位点口袋中的靶碱基的结合需要蛋氨酸。我们的数据表明，DPPY基序中的酪氨酸残基保守的DNA-（腺嘌呤-N6）-MTases的活性位点堆叠到翻转的靶碱基。用丙氨酸取代DPPY基序的天冬氨酸残基消除了碱基翻转，表明该残基接触并稳定了翻转的碱基。Ser 188位于一个环旁边的活性中心由丙氨酸的交换导致了一个七至八倍的减少k（翻转），这也减少了与基板具有改变GATC识别位点和在没有的cDNAMet。这些发现提供的证据表明，酶主动启动基地翻转稳定的过渡状态的过程。在含有非规范序列中的靶碱基的底物中碱基翻转的速率降低表明，MTase的DNA识别在碱基翻转之前开始。DNA识别、辅因子结合和碱基翻转是相关的，并且只有当酶结合到同源靶位点并且可获得C3 Met时才发生有效的碱基翻转。(C)2004爱思唯尔有限公司保留所有权利。
By stopped-flow kinetics using 2-aminopurine as a probe to detect base flipping, we show here that base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase (MTase) is a biphasic process: target base flipping is very fast (k(flip) > 240 s(-1)), but binding of the flipped base into the active site pocket of the enzyme is slow (k = 0.1-2 s(-1)). Whereas base flipping occurs in the absence of S-adenosyl-L-methionine (AdoMet), binding of the target base in the active site pocket requires AdoMet. Our data suggest that the tyrosine residue in the DPPY motif conserved in the active site of DNA-(adenine-N6)-MTases stacks to the flipped target base. Substitution of the aspartic acid residue of the DPPY motif by alanine abolished base flipping, suggesting that this residue contacts and stabilizes the flipped base. The exchange of Ser188 located in a loop next to the active center by alanine led to a seven- to eightfold reduction of k(flip), which was also reduced with substrates having altered GATC recognition sites and in the absence of AdoMet. These findings provide evidence that the enzyme actively initiates base flipping by stabilizing the transition state of the process. Reduced rates of base flipping in substrates containing the target base in a non-canonical sequence demonstrate that DNA recognition by the MTase starts before base flipping. DNA recognition, cofactor binding and base flipping are correlated and efficient base flipping takes place only if the enzyme has bound to a cognate target site and AdoMet is available. (C) 2004 Elsevier Ltd. All rights reserved.