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首页> 外文期刊>Journal of Molecular Biology >Stopped-flow and mutational analysis of base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase.
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Stopped-flow and mutational analysis of base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase.

机译：大肠杆菌Dam DNA-（腺嘌呤-N6）-甲基转移酶对碱基翻转的停止流动和突变分析。

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摘要

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.
机译：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，我们在此处显示了大肠杆菌Dam DNA-（腺嘌呤-N6）-甲基转移酶（MTase）的碱基翻转是一个双相过程：目标碱基翻转非常快速度快（k（flip）> 240 s（-1）），但是将翻转后的碱基结合到酶的活性位点口袋中的速度很慢（k = 0.1-2 s（-1））。在没有S-腺苷-1-甲硫氨酸（AdoMet）的情况下发生碱基翻转，而在活性位点口袋中目标碱基的结合则需要AdoMet。我们的数据表明，DPPY基序中的酪氨酸残基在DNA-（腺嘌呤-N6）-MTases的活性位点中保守，堆积到翻转的靶碱基上。丙氨酸取代DPPY基序的天冬氨酸残基消除了碱基的翻转，表明该残基接触并稳定了翻转的碱基。丙氨酸位于靠近活性中心的环路中的Ser188的交换导致k（flip）降低了7到8倍，这在底物的GATC识别位点改变且没有AdoMet的情况下也降低了。这些发现提供了证据，表明该酶通过稳定过程的过渡态而主动引发碱基翻转。在非规范序列中包含靶碱基的底物中碱基翻转的速率降低，表明MTase对DNA的识别在碱基翻转之前就开始了。 DNA识别，辅因子结合和碱基翻转是相关的，只有在酶已结合到同源靶位并且AdoMet可用的情况下，才能进行有效的碱基翻转。

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