ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

抗生素耐药性的酶学

• 批准号: 6831181
• 负责人: RICHARD N ARMSTRONG
• 金额: $ 22.65万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6831181
• 关键词: ENZYMOLOGY; ANTIBIOTIC; RESISTANCE

EXCEEDTHE SPACE PROVIDED. In the last two decades it has become increasingly clear that the efficacy of antibiotics for the treatment of infectious diseases is in jeopardy due to the common appearance of drug resistant strains of microorganisms. Fosfomycin is a potent, broad-spectrum antibiotic effective against both Gram-positive and Gram-negative microorganisms. A decade after its introduction plasmid-mediated resistance to fosfomycin was observed in the clinic. Investigations supported by this project have established that the resistance is due to a metalloenzyme (FosA) that catalyzes the addition ofglutathione to the antibiotic, rendering it inactive. Moreover, similar resistance elements have now been shown to exist in the genomes of several pathogenic microorganisms including, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus anthrasis, Brucella melitensis, and Listeria monocytogenes. Genomic and biochemical analysis suggest that there are three distinct subgroups of metalloenzymes, termed FosA, FosB and FosX, that confer resistance through somewhat different chemical mechanisms. The objectives of this research project are to identify plasmid and genomically encoded proteins involved in microbial resistance to the antibiotic fosfomycin and to elucidate the underlying structural and mechanistic enzymology of resistance. These objectives will be accomplished by integrating enzymological, biophysical and genomic analyses of the resistance problem. The three-dimensional structures of the FosA homologue PA 1129 from Pseudomonas aeruginosa and its close relatives will be determined X-ray crystallography and mass spectrometry. The chemical and kinetic mechanisms of catalysis will be elucidated by: (i) examination of the inner coordination sphere of Mn 2+in FosA by EPR and ENDOR spectroscopy; (ii) a steady state kinetic analysis of the thiol selectivity of FosA and FosB and the inhibition of these enzymes by phosphonoformate with particular emphasis on the enzymes from the pathogens Pseudomonas aeruginosa and Staphylococcus aureus; and (iii) a mechanistic study of the unique hydration reaction catalyzed by FosX. The thermodynamics and molecular dynamics of the interaction of metals, substrates and inhibitors with FosA and FosB will be examined by: (i) a determination of the thermodynamics of binding of metals, substrate and inhibitor ligands by isothermal titration calorimetry and; (ii) an NMR investigation of the changes in the molecular dynamics of the protein on metal and substrate binding to assess the contribution of high-frequency motions to the overall binding energetics. The intent of these investigation is to establish the mechanistic and structural bases for the design of drugs to counter both plasmid borne and genomically encoded resistance to fosfomycin. PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。在过去的二十年中，越来越清楚的是，由于微生物的耐药菌株的普遍出现，抗生素用于治疗感染性疾病的功效处于危险之中。磷霉素是一种有效的广谱抗生素，对革兰氏阳性和革兰氏阴性微生物都有效。在其引入十年后，在临床上观察到质粒介导的对磷霉素的耐药性。由该项目支持的调查已经确定，耐药性是由于金属酶（FosA）催化谷胱甘肽加入抗生素，使其失活。此外，类似的抗性元件现已显示存在于几种病原微生物的基因组中，包括铜绿假单胞菌、金黄色葡萄球菌、炭疽杆菌、羊种布鲁氏菌和单核细胞增生李斯特菌。基因组和生化分析表明，有三个不同的金属酶亚组，称为FosA，FosB和FosX，通过不同的化学机制赋予抗性。本研究项目的目的是鉴定质粒和基因组编码的蛋白质参与微生物耐药性的抗生素磷霉素，并阐明潜在的结构和机制的耐药性酶学。这些目标将通过整合抗性问题的酶学、生物物理和基因组分析来实现。来自铜绿假单胞菌及其近亲的FosA同源物PA 1129的三维结构将通过X射线晶体学和质谱法确定。通过EPR和ENDOR光谱分析FosA中Mn 2+的内配位圈，稳态动力学分析FosA和FosB的硫醇选择性以及膦甲酸酯对这些酶的抑制作用，特别是对铜绿假单胞菌和金黄色葡萄球菌的抑制作用，阐明了催化作用的化学和动力学机理;以及（iii）FosX催化的独特水合反应机理研究。金属、底物和抑制剂与FosA和FosB相互作用的热力学和分子动力学将通过以下方法进行研究：（i）通过等温滴定量热法测定金属、底物和抑制剂配体结合的热力学;（ii）对蛋白质在金属和底物结合上的分子动力学变化的NMR研究，以评估高-频率运动到整体结合能。这些研究的目的是建立机制和结构基础的药物设计，以对抗质粒携带和基因组编码的耐药性磷霉素。性能现场=