ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

EXCEED提供的空间。在过去的二十年里，越来越清楚的是，由于耐药微生物菌株的常见出现，抗生素治疗传染病的疗效正处于危险之中。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细菌都有效。在其引入十年后，在临床上观察到了对磷霉素的质粒介导的耐药性。该项目支持的研究已经确定，耐药性是由于一种金属酶(FOSA)催化谷胱甘肽加成到抗生素中，使其失去活性。此外，目前已证明在铜绿假单胞菌、金黄色葡萄球菌、炭疽杆菌、羊布鲁氏菌和单核细胞增生性李斯特菌等几种病原微生物的基因组中存在类似的耐药元件。基因组和生化分析表明，有三种不同的金属酶亚群，分别命名为FOSA、FOSB和FosX，它们通过不同的化学机制赋予抗性。本研究项目的目的是鉴定与微生物对磷霉素耐药有关的质粒和基因编码蛋白，并阐明耐药的潜在结构和机制。这些目标将通过整合抗药性问题的酶学、生物物理和基因组分析来实现。对来自铜绿假单胞菌及其近亲的FOSA同系物PA 1129的三维结构进行了X射线结晶学和质谱学测定。(I)用EPR和Endor光谱分析FOSA中Mn2+的内部配位范围；(Ii)稳态动力学分析FOSA和FOSB的硫醇选择性以及磷甲酸盐对这些酶的抑制作用，重点是铜绿假单胞菌和金黄色葡萄球菌的酶；(Iii)FOX催化独特的水合反应的机理研究。金属、底物和缓蚀剂与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)用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)核磁共振研究蛋白质与金属和底物结合的分子动力学变化，以评估高频运动对整体结合能的贡献。这些研究的目的是为药物的设计建立机制和结构基础，以对抗磷霉素携带的和基因编码的耐药性。表演网站========================================Section End===========================================

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===========================================