Department of Biochemistry, Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec H3G 0B1, Canada.
J Biol Chem. 2012 Apr 13;287(16):13094-102. doi: 10.1074/jbc.M112.349670. Epub 2012 Feb 24.
Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.
通过一类被称为氨基糖苷 O-磷酸转移酶(APHs)的酶进行酶促磷酸化是细菌对抗氨基糖苷类抗生素产生抗性的主要机制。APH(2″)亚家族的成员由于在致病菌株中的普遍性及其广泛的底物谱而具有特殊的临床意义。APH(2″)酶对 ATP 或 GTP 作为磷酸供体显示出不同的偏好,其中氨基糖苷 2″-磷酸转移酶 IVa(APH(2″)-IVa)是一种能够以相当的效率利用这两种核苷酸的成员。我们在此报告了 APH(2″)-IVa 的四个晶体结构,其中两个是野生型酶,两个是单个氨基酸突变体,每个都与腺苷或鸟苷复合物。这些结构共同提供了对该酶核苷结合位点结构的详细了解,并揭示了赋予双重核苷酸特异性的关键元素,包括核苷结合口袋内部的溶剂网络和结构域间连接环的构象。稳态动力学研究,以及与 APH(2″)亚家族和其他氨基糖苷激酶成员的序列和结构比较,合理地解释了这些酶对不同底物的偏好。最后,尽管整体序列相似性和结构同源性较差,但对 APH(2″)-IVa 的核苷结合口袋的分析显示出与真核酪蛋白激酶 2(CK2)惊人的相似性,CK2 也表现出双重核苷酸特异性。这些结果,再加上针对 CK2 的大量现有抑制剂,可作为针对临床相关 APH 酶的核苷酸竞争性抑制剂设计的结构基础。