Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region.
Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region.
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DOI：
10.1093/nar/gkn460
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发表时间：
2008-08
影响因子：
14.9
通讯作者：
Tse-Dinh, Yuk-Ching
中科院分区：
文献类型：
--
作者：
Sorokin, Elena P.;Cheng, Bokun;Rathi, Siddarth;Aedo, Sandra J.;Abrenica, Maria V.;Tse-Dinh, Yuk-Ching
在细菌拓扑异构酶I中，在亲核酪氨酸旁边的活性位点发现保守的甲硫氨酸残基。在重组鼠疫耶尔森氏菌拓扑异构酶I（YTOP）中用精氨酸取代该甲硫氨酸残基是在该位置发现的唯一取代，其在大肠杆菌中诱导SOS反应。M326 R突变体YTOP的过表达导致存活力的104个对数损失。对纯化的Y.鼠疫和E.大肠杆菌突变体拓扑异构酶I表明，Met到Arg取代影响催化循环的DNA再连接步骤。在突变体E的活性部位区域引入额外的正电荷。coli拓扑异构酶I活性改变了最佳活性的pH，并降低了Mg 2+结合亲和力。这项研究表明，TOPRIM基序外的取代，其直接结合Mg 2+，但可以抑制Mg 2+结合和DNA的酶的再连接，增加共价切割复合物的积累，具有杀菌效果。能够特异性抑制Mg ~（2+）依赖的细菌拓扑异构酶I再连接的小分子化合物可以开发成有用的新型抗菌化合物。这种方法将类似于在抗病毒治疗中通过HIV整合酶抑制二价离子依赖性链转移。
Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in ∼4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg2+ binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg2+directly, can nonetheless inhibit Mg2+ binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg2+ dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy.
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