Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montréal, Québec Canada, H3G 1Y6.
Centre de Recherche en Biologie Structurale, McGill University, Bellini Life Science Complex, 3649 Promenade Sir William Osler, Montréal, Québec Canada, H3G 0B1.
ACS Chem Biol. 2020 Mar 20;15(3):686-694. doi: 10.1021/acschembio.9b00904. Epub 2020 Mar 6.
Aminoglycoside antibiotics have lost much of their effectiveness due to widespread resistance, primarily via covalent modification. One of the most ubiquitous enzymes responsible for aminoglycoside resistance is aminoglycoside -nucleotidyltransferase(2″), which catalyzes a nucleotidylation reaction. Due to its clinical importance, much research has focused on dissecting the mechanism of action, some of it dating back more than 30 years. Here, we present structural data for catalytically informative states of the enzyme, i.e., ANT(2″) in complex with adenosine monophosphate (AMP) and tobramycin (inactive-intermediate state) and in complex with adenylyl-2″-tobramycin, pyrophosphate, and Mn(product-bound state). These two structures in conjunction with our previously reported structure of ANT(2″)'s substrate-bound complex capture clinical states along ANT(2″)'s reaction coordinate. Additionally, isothermal titration calorimetry (ITC)-based studies are presented that assess the order of substrate binding and product release. Combined, these results outline a kinetic mechanism for ANT(2″) that contradicts what has been previously reported. Specifically, we show that the release of adenylated aminoglycoside precedes pyrophosphate. Furthermore, the ternary complex structures provide additional details on the catalytic mechanism, which reveals extensive similarities to the evolutionarily related DNA polymerase-β superfamily.
由于广泛的耐药性，氨基糖苷类抗生素的功效大大降低，主要是通过共价修饰。导致氨基糖苷类抗生素耐药性的最常见的酶之一是氨基糖苷类-核苷酸转移酶(2″)，它催化核苷酸化反应。由于其临床重要性，许多研究都集中在剖析作用机制上，其中一些研究可以追溯到 30 多年前。在这里，我们呈现了具有催化信息的酶结构数据，即与腺苷一磷酸(AMP)和妥布霉素(无活性中间体状态)结合的 ANT(2″)，以及与腺苷酰基-2″-妥布霉素、焦磷酸和 Mn(产物结合状态)结合的 ANT(2″)。这两个结构结合我们之前报道的 ANT(2″)的底物结合复合物的结构，捕获了沿着 ANT(2″)反应坐标的临床状态。此外，还提出了基于等温滴定量热法(ITC)的研究，评估了底物结合和产物释放的顺序。综合这些结果，概述了 ANT(2″)的动力学机制，这与之前报道的内容相矛盾。具体来说，我们表明，被腺苷酸化的氨基糖苷类的释放先于焦磷酸盐。此外，三元复合物结构为催化机制提供了更多细节，这揭示了与进化上相关的 DNA 聚合酶-β超家族的广泛相似性。
