Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9 from Escherichia coli
Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9 from Escherichia coli
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DOI：
10.1042/bj3340387
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发表时间：
1998-09-01
影响因子：
4.1
通讯作者：
Kleanthous, C
中科院分区：
文献类型：
--
作者：
Pommer, AJ;Wallis, R;Kleanthous, C
细菌毒素大肠杆菌素E9的细胞毒性是由于一种非特异性DNA酶穿透受感染生物体的细胞质并导致细胞死亡。我们报告的第一个酶学特性的过表达和纯化的15 kDa的DNA酶域（E9 DNA酶）从这类毒素。CD光谱显示E9 DNA酶在溶液中是结构化的，并且分析超离心数据表明该酶是单体。通过使用以小牛胸腺DNA作为底物的分光光度测定法，将E9 DNA酶的核酸酶活性与充分研究的非特异性DNA酶I进行比较。这两种酶都需要二价金属离子才能活性，但与DNA酶I不同，E9 DNA酶不被Ca 2+离子激活。有些令人惊讶的是，E9 DNA酶在过渡金属如Ni 2+和Co 2+存在下显示出最佳活性和线性动力学，但在金属如Mg 2+和Ca 2+存在下显示出非线性动力学。相反，Ni 2+和其他过渡金属在基于质粒的切口测定中表现出较差的活性，产生大量的线性化质粒，而Mg 2+非常活跃，主要中间体是开环DNA。结果表明，在进入细菌细胞，E9 DNA酶很可能主要表现出Mg2+依赖性的切割活性对染色体DNA，虽然其他金属也可以用来引入单链和双链裂解。
The cytotoxicity of the bacterial toxin colicin E9 is due to a nonspecific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structured in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was compared with the well-studied, non-specific DNase I by using a spectrophotometric assay with calf thymus DNA as the substrate. Both enzymes require divalent metal ions for activity but, unlike DNase I, the E9 DNase is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transition metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition metals showed poor activity in a plasmid-based nicking assay, yielding significant amounts of linearized plasmid, whereas Mg2+ was very active, with the main intermediate being open-circle DNA. The results suggest that, on entry into bacterial cells, the E9 DNase is likely to exhibit primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.