Green Keith D, Biswas Tapan, Chang Changsoo, Wu Ruiying, Chen Wenjing, Janes Brian K, Chalupska Dominika, Gornicki Piotr, Hanna Philip C, Tsodikov Oleg V, Joachimiak Andrzej, Garneau-Tsodikova Sylvie
⊥Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40536-0596, United States.
∇Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne, Illinois 60439, United States.
Biochemistry. 2015 May 26;54(20):3197-206. doi: 10.1021/acs.biochem.5b00244. Epub 2015 May 12.
Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.
增强型细胞内存活（Eis）家族的蛋白质是多功能乙酰转移酶，可在几种氨基糖苷类（AGs）的多个位置将胺乙酰化。它们的上调赋予耐药性。Eis的同源物存在于多种细菌中，包括许多病原体。结核分枝杆菌的Eis（Eis_Mtb）已得到充分表征。在本研究中，我们探索了炭疽芽孢杆菌Eis家族蛋白（Eis_Ban）的AG特异性和催化效率。对六种AGs乙酰化特异性和催化效率的动力学分析表明，Eis_Ban在底物结合和催化效率方面与Eis_Mtb均存在显著差异。几种AGs的乙酰化胺数量也不同，表明Eis_Ban具有独特的区域特异性。此外，最近发现的大多数Eis_Mtb抑制剂对Eis_Ban没有抑制作用，凸显了这两种酶之间的差异。为了解释这些差异，我们确定了Eis_Ban的晶体结构。Eis_Ban和Eis_Mtb晶体结构的比较表明，它们各自底物结合口袋周围的关键残基有很大差异，这解释了它们不同的特异性。我们的结果表明，Eis家族的乙酰转移酶在进化过程中发生了分歧，以获得不同的特异性，同时保持催化效率，这可能是为了应对不同的化学挑战。这些酶独特的特异性特征可作为开发具有新型修饰的AGs的工具，并有助于指导特定的AG治疗以避免Eis介导的耐药性。
