Engineering a nicking endonuclease N.AlwI by domain swapping
Engineering a nicking endonuclease N.AlwI by domain swapping
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DOI：
10.1073/pnas.241215698
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发表时间：
2001-11-06
影响因子：
11.1
通讯作者：
Kong, HM
中科院分区：
文献类型：
--
作者：
Xu, Y;Lunnen, KD;Kong, HM
通过基因工程改变酶的功能对分子生物学家来说仍然是一个挑战。在这里我们展示一个例子，其中改变一种酶的寡聚化状态会改变其功能。II型限制内切酶，如AlwI，通常折叠成两个独立的结构域：一个DNA结合结构域和一个催化/二聚化结构域。我们将AlwI的假定二聚化结构域与来自一种切口酶N.BstNBI的无功能二聚化结构域进行了替换。所得的嵌合酶N.AlwI不再形成二聚体。有趣的是，单体形式的N.AlwI仍然识别与AlwI相同的序列，但仅切割含有5'-GGATC - 3'序列的DNA链（上链）。相比之下，野生型AlwI在溶液中以二聚体形式存在并切割两条DNA链；上链被结合到该序列的一种酶切割，其互补的下链被与第一种酶二聚化的第二种酶切割。N.AlwI无法形成二聚体，因此作为单体切割DNA。此外，这种经过改造的切口酶至少与野生型AlwI一样有活性，因此是一种有用的酶。据我们所知，这是通过结构域替换创造一种切口酶的首次报道。
Changing enzymatic function through genetic engineering still presents a challenge to molecular biologists. Here we present an example in which changing the oligomerization state of an enzyme changes its function. Type Ils restriction endonucleases such as AlwI usually fold into two separate domains: a DNA-binding domain and a catalytic/dimerization domain. We have swapped the putative dimerization domain of AlwI with a nonfunctional dimerization domain from a nicking enzyme, N.BstNBI. The resulting chimeric enzyme, N.AlwI, no longer forms a dimer. Interestingly, the monomeric N.AlwI still recognizes the same sequence as AlwI but only cleaves the DNA strand containing the sequence 5'-GGATC-3' (top strand). In contrast, the wild-type AlwI exists as a dimer in solution and cleaves two DNA strands; the top strand is cleaved by an enzyme binding to that sequence, and its complementary bottom strand is cleaved by the second enzyme dimerized with the first enzyme. N.AlwI is unable to form a dimer and therefore nicks DNA as a monomer. In addition, the engineered nicking enzyme is at least as active as the wild-type AlwI and is thus a useful enzyme. To our knowledge, this is the first report of creating a nicking enzyme by domain swapping.