Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons):: a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase
Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons):: a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase
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DOI：
10.1128/aac.48.5.1856-1864.2004
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发表时间：
2004-05-01
影响因子：
4.9
通讯作者：
Gross, CH
中科院分区：
文献类型：
--
作者：
Bellon, S;Parsons, JD;Gross, CH
拓扑异构酶IV和DNA促旋酶是相关的细菌II型拓扑异构酶，其利用来自ATP水解的自由能来催化细菌基因组中的拓扑变化。DNA促旋酶的基本功能是将负DNA超螺旋引入基因组中，而拓扑异构酶IV的基本功能是在复制后使子染色体去连锁。在这里，我们报告的晶体结构的43 kDa的N-末端片段的大肠杆菌拓扑异构酶IV帕雷亚基复合腺苷酰亚胺二磷酸在2.0埃分辨率和24 kDa的N-末端片段的帕雷亚基复合新生霉素在2.1埃分辨率。解析的帕雷结构与已知的促旋酶B（GyrB）亚基结构惊人地相似。我们还鉴定了帕雷（M74）和GyrB（I78）中的单一位置等同氨基酸残基，当交换时，新生霉素对拓扑异构酶IV的效力增加近20倍（至12 nM）。相应的促旋酶（I78 M）交换使新生霉素的效力降低20倍（至1.0 μ M）。这些数据为新生霉素对拓扑异构酶IV的效力显著低于对DNA促旋酶的效力的观察提供了解释。此外，酶动力学参数也受到影响。在促旋酶中，ATP的Km值增加近5倍，Vmax值下降近30%。与此相反，拓扑异构酶IV ATP K-m降低了6倍，V-max增加了约2倍，从野生型值。这些数据表明帕雷M74和GyrB 178侧链对酶的底物亲和力和催化效率产生相反的影响。
Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-Angstrom resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-Angstrom resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 muM). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K-m increased approximate to5-fold and the V-max decreased approximate to30%. In contrast, the topoisomerase IV ATP K-m decreased by a factor of 6, and the V-max increased approximate to2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB 178 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.