Asp-to-Asn substitution at the first position of the DxD TOPRIM motif of recombinant bacterial topoisomerase I is extremely lethal to E. coli.
Asp-to-Asn substitution at the first position of the DxD TOPRIM motif of recombinant bacterial topoisomerase I is extremely lethal to E. coli.
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DOI：
10.1016/j.jmb.2008.10.073
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发表时间：
2009-01-16
影响因子：
5.6
通讯作者：
Tse-Dinh, Yuk-Ching
中科院分区：
文献类型：
--
作者：
Cheng, Bokun;Annamalai, Thirunavukkarasu;Sorokin, Elena;Abrenica, Maria;Aedo, Sandra;Tse-Dinh, Yuk-Ching
在许多核苷酸转移酶中发现的TOPRIM结构域包含一个DxD基序，该基序参与了镁离子的催化配位。鼠疫耶尔森氏菌拓扑异构酶I(YpTOP)基序第一个天冬氨酸残基(D117N)的天冬氨酸到天冬氨酸(Asn)替换的中高拷贝数质粒克隆在没有第二位点突变的情况下，即使在严格调控的不良启动子的控制下，并用2%的葡萄糖抑制表达，也不能在大肠杆菌中产生。阿拉伯糖诱导整合到染色体上的单拷贝YpTOP-D117N突变基因在2.5h内使细胞杀伤率达到105倍，试图诱导相应的大肠杆菌拓扑异构酶I突变体(EcTOP-D111N)在高拷贝数质粒上表达会导致活性丧失或克隆回复到野生型。YpTOP-D119N和EcTOP-D113N的高拷贝克隆在TOPRIM基序的第二个Asp处有ASN替换，可以稳定保持，但过表达也会显著降低细胞存活率。这些TOPRIM残基上的天冬氨酸到天冬氨酸的取代可以选择性地降低镁离子的结合亲和力，同时对活性部位的几何结构造成最小的破坏，导致与切割的DNA的共价复合物被捕获，并导致细菌细胞死亡。TOPRIM第一个位置的极端敏感性表明，这可能是一个有用的结合小分子的位置，这些小分子可以作为拓扑异构酶毒物。
The TOPRIM domain found in many nucleotidyl transferases contains a DxD motif involved in magnesium ion coordination for catalysis. Medium to high copy number plasmid clones of Yersinia pestis topoisomerase I (YpTOP) with Asp to Asn substitution at the first aspartate residue (D117N) of this motif could not be generated in Escherichia coli without second site mutation even when expression was under the control of the tightly regulated BAD promoter and suppressed by 2% glucose in the medium. Arabinose induction of a single copy YpTOP-D117N mutant gene integrated into the chromosome resulted in ~105 fold of cell killing in 2.5 h. Attempt to induce expression of the corresponding E. coli topoisomerase I mutant (EcTOP-D111N) encoded on a high copy number plasmid resulted in either loss of viability or reversion of the clone to wild-type. High copy plasmid clones of YpTOP-D119N and EcTOP-D113N with the Asn substitution at the second Asp of the TOPRIM motif could be stably maintained, but overexpression also decreased cell viability significantly. The Asp to Asn substitutions at these TOPRIM residues can selectively decrease Mg2+ binding affinity with minimal disruption of the active site geometry, leading to trapping of the covalent complex with cleaved DNA and causing bacterial cell death. The extreme sensitivity of the first TOPRIM position suggested that this might be a useful site for binding of small molecules that could act as topoisomerase poisons.
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