Aminoglycoside Resistance in Bacteria

描述（由申请人提供）：双功能氨基糖苷类修饰酶AAC（6“）-Ie-APH（2”）-Ia是革兰氏阳性球菌中氨基糖苷类抗生素的最重要的耐药酶，因为它单独产生对几乎所有氨基糖苷类抗生素的耐药性，并在临床肠球菌和葡萄球菌分离株中广泛传播。在革兰氏阴性菌中出现密切相关的单功能APH（2”）-Ib磷酸转移酶和AAC（6“）-Im乙酰转移酶，预示着这些广谱酶在多种临床重要病原体中传播的潜力。缺乏有关这些酶的动力学和结构信息构成了设计新型氨基糖苷类抗生素和氨基糖苷类修饰酶抑制剂的主要障碍。长期目标是阐明氨基糖苷类修饰酶与其底物相互作用的动力学和结构机制，为合理设计新的有效抗生素提供有力依据。本特定申请的目的是通过追求三个具体目标对APH（2”）-Ib、AAC（6 '）-Im和AAC（6'）-Ie-APH（2”）-Ia酶进行详细研究：进行AAC（6 '）-Im、APH（2”）-Ib和AAC（6'）-Ie-APH（2”）-Ia氨基糖苷修饰酶的动力学和结构研究; 2）阐明氨基糖苷磷酸转移酶对核苷酸三磷酸选择性的分子机制; 3）评价双功能AAC（6 '）-Ie-APH（2”）-Ia氨基糖苷修饰酶的进化潜力。多学科的方法，包括分子生物学，蛋白质化学，详细的酶学，纯化和结构分析的反应产物的NMR和质谱，和X-射线晶体学的天然酶及其复合物的产品，底物和底物类似物将被用来阐明这些临床上重要的耐药酶的机制方面。本资助申请中公开的AAC（6 ′）-Im、APH（2 ″）-Ib和AAC（6 ′）-Ie-APH（2 ″）-Ia酶的详细结构和动力学研究以及氨基糖苷磷酸转移酶识别核苷酸三磷酸（NTP）的分子机制的阐明将提供对它们独特的广泛活性的深入了解。这些研究也将有助于我们了解这些酶的作用机制，并为合理设计新型抗氨基糖苷类修饰酶修饰的氨基糖苷类抗生素和针对氨基糖苷类磷酸转移酶核苷酸结合位点的抑制剂提供坚实的基础。对双功能AAC（6 '）-Ie-APH（2”）-Ia酶的进化潜力的评估将为临床上重要的氨基糖苷类抗生素的未来应用提供重要的指导。最终，这些知识将为设计新的氨基糖苷类抗生素和氨基糖苷类修饰酶的抑制剂以治疗危及生命的感染提供基础。

DESCRIPTION (provided by applicant): The bifunctional aminoglycoside-modifying enzyme AAC(6')-Ie-APH(2")-Ia is the single most important resistance enzyme for aminoglycoside antibiotics in Gram-positive cocci as it is singlehandedly produces resistance to virtually all aminoglycoside antibiotics and broadly disseminated among clinical enterococcal and staphylococcal isolates. Appearance of the closely-related monofunctional APH(2")-Ib phosphotransferase and AAC(6')-Im acetyltransferase in Gram-negative bacteria heralding the potential for spread of these expanded- spectrum enzymes among a wide variety of clinically important pathogens. Lack of kinetic and structural information regarding these enzymes constitutes the major impediment for the design of novel aminoglycoside antibiotics and inhibitors of aminoglycoside-modifying enzymes. The long-term goal is to delineate kinetic and structural mechanisms of interaction of aminoglycoside-modifying enzymes with their substrates to provide strong basis for the rational design of new effective antibiotics. The objectives in this particular application is to conduct detailed studies of the APH(2")-Ib, AAC(6')-Im, and AAC(6')-Ie-APH(2")-Ia enzymes by pursuing three specific aims: 1. Perform kinetics and structural studies of the AAC(6')-Im, APH(2")-Ib, and AAC(6')-Ie- APH(2")-Ia aminoglycoside-modifying enzymes; 2) Elucidate molecular mechanisms for the nucleotide triphosphate selectivity by the aminoglycoside phosphotransferases; and 3) Evaluate evolutionary potential of the bifunctional AAC(6')-Ie-APH(2")-Ia aminoglycoside-modifying enzyme. Multidisciplinary approach that include molecular biology, protein chemistry, detailed enzymology, purification and structural analyses of the products of the reactions by NMR and mass spectrometry, and X-ray crystallography of the native enzymes and their complexes with products, substrates and substrate analogues will be utilized to elucidate mechanistic aspects of these clinically important antibiotic-resistance enzymes. The detailed structural and kinetic studies of the AAC(6')-Im, APH(2")-Ib, and AAC(6')-Ie-APH(2")-Ia enzymes and elucidation of the molecular mechanism of recognition of nucleotide triphosphates (NTPs) by aminoglycoside phosphotransferases disclosed in this grant proposal will provide insight into their unique over-encompassing breadth of activity. These studies will also contribute to our understanding of the mechanisms of these enzymes, and provide solid basis for the rational design of novel aminoglycoside antibiotics resistant to modification by the aminoglycoside-modifying enzymes and inhibitors targeting the nucleotide binding site of aminoglycoside phosphotransferases. Evaluation of the evolutionary potential of the bifunctional AAC(6')-Ie-APH(2")-Ia enzyme towards broadening the spectrum of conferred resistance to aminoglycoside antibiotics would provide an important guidance for the future utility of clinically important aminoglycosides. Ultimately, such knowledge will provide the basis for the design of new aminoglycoside antibiotics and inhibitors of the aminoglycoside- modifying enzymes for treatment of life-threatening infections.