STRUCTURAL STUDIES ON AMINOGLYCOSIDE PHOSPHOTRANSFERASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 抗生素耐药性产生的一个主要因素是细菌酶的存在，这种酶可以对常见的抗生素进行化学修饰。氨基糖苷类药物(如卡那霉素、妥布霉素和庆大霉素)是目前几乎普遍耐药的一类抗菌药物。肠球菌对庆大霉素的高水平耐药是由APH(2？)使用三磷酸腺苷作为辅基，在抗生素上的特定羟基发生磷酸化的酶的亚家族。了解这些酶是如何结合和失活氨基糖苷类的，将为设计这些酶的特定抑制剂提供有价值的信息。我们正在研究所有四种APH(2？)来自粪肠球菌的APH(2？)-Ia和APH(2？)-IIa，来自鸡肠球菌的APH(2？)-IIa，来自酪蛋白肠球菌的APH(2？)-Iva。报道了APH(2？)-IIa的庆大霉素二元络合物和AMPPCP-链霉素三元络合物的三维结构，并报道了APH(2？)-IIIa和APH(2？)-IVa的结晶条件。APH(2？)-IIa和APH(2？)-IVa结构均以APH(2？)-IIa为搜索模型，用分子置换方法求解。此外，还从一种SeMet APH(2？)-Ia晶体中收集了三个波长的MAD数据，并对其结构进行了求解和部分精化。关于这些酶的二元和三元络合物的其他结构研究正在进行中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major factor in the emergence of antibiotic resistance is the existence of bacterial enzymes that chemically modify common antibiotics. One such family of anti-bacterials to which there is now almost universal resistance are the aminoglycosides (e.g. kanamycin, tobramycin and gentimicin). High level resistance to gentamicin in enterococci is mediated by a group of four phosphotransferases belonging to the APH(2?) sub-family of enzymes which phosphorylate at a specific hydroxyl group on the antibiotic, using ATP as a cosubstrate. An understanding of how these enzymes bind and deactivate the aminoglycosides will provide valuable information for the design of specific inhibitors of these enzymes. We are studying all four of the APH(2?) phosphotransferases, APH(2?)-Ia and APH(2?)-IIa from Enterococcus faecium, APH(2?)-IIIa from E. gallinarum, and APH(2?)-IVa from E. casseliflavus. The three dimensional structures of the binary gentamicin complex and a ternary AMPPCP-streptomycin complex of APH(2?)-IIa has been determined and recently published and the other crystallization conditions of APH(2?)-IIIa and APH(2?)-IVa have been reported. Both the APH(2?)-IIIa and APH(2?)-IVa structures has been solved by molecular replacement using APH(2?)-IIa as the search model. In addition, three wavelength MAD data has been collected from a SeMet APH(2?)-Ia crystal and the structure has been solved and partially refined. Additional structural studies on binary and ternary complexes of these enzymes are underway.