ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

超出规定的空间。在过去二十年中，越来越清楚的是，抗生素治疗传染病的效力由于普遍出现的耐药微生物菌株而受到威胁。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。引入磷霉素十年后，在临床观察到质粒介导的对磷霉素的耐药性。该项目支持的研究已经确定，耐药性是由于一种金属酶（FosA）催化向抗生素中添加谷胱甘肽，使其失去活性。此外，类似的耐药元件现在已被证明存在于几种致病微生物的基因组中，包括铜绿假单胞菌、金黄色葡萄球菌、炭疽芽孢杆菌、布鲁氏菌和单核增生李斯特菌。基因组和生化分析表明，金属酶有三个不同的亚群，称为FosA、FosB和fox，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参与微生物对磷霉素耐药性的质粒和基因组编码蛋白，并阐明耐药性的潜在结构和机制酶学。这些目标将通过整合对抗性问题的酶学、生物物理和基因组分析来实现。铜绿假单胞菌及其近缘菌FosA同源物pa1129的三维结构将用x射线晶体学和质谱法测定。(1)利用EPR和ENDOR光谱对FosA中Mn 2+的内配位球进行了研究；（ii）对FosA和FosB的巯基选择性和膦甲酸酯对这些酶的抑制作用进行稳态动力学分析，特别强调了铜绿假单胞菌和金黄色葡萄球菌的酶；(3)对FosX催化的独特水化反应进行机理研究。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式进行研究：(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进行核磁共振研究，以评估高频运动对整体结合能量的贡献。这些研究的目的是建立机制和结构基础的药物设计对抗质粒携带和基因编码的磷霉素耐药。网站性能 ======================================== 节结束 ===========================================

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