ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

在过去的二十年里，越来越清楚的是，抗生素治疗 由于耐药微生物菌株的普遍出现，传染病处于危险之中。 了解抗菌素耐药性的机制对于临床上有效的患者护理至关重要， 这对于制定战略以加强对故意传播病原体的生物防御至关重要。 磷霉素是一种有效的广谱抗生素，对革兰氏阳性菌和革兰氏阴性菌均有效 微生物的在其引入十年后，在大肠杆菌中观察到质粒介导的磷霉素抗性。 诊所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，使其失去活性。类似的抵抗分子 现已证明存在于几种病原微生物的基因组中，包括假单胞菌 铜绿假单胞菌、金黄色葡萄球菌、炭疽杆菌、羊种布鲁氏菌、单核细胞增生李斯特菌和 肉毒梭菌。该项目的基因组和生化分析表明，有三种不同的 金属酶的亚群，称为FosA，FosB和FosX，通过某种程度上不同的方式赋予抗性， 化学机制。本研究项目的目标是鉴定质粒和基因组编码的 蛋白质参与微生物耐磷霉素和阐明潜在的结构和机制， 抗性酶学这些目标将通过整合酶学，生物物理学和 对抗药性问题的基因组分析。假单胞菌FosA的三维结构 铜绿假单胞菌及其相关物FosB和FosX的含量将通过X射线晶体学测定。化学和 催化的内在机制将通过以下方式阐明：（i）检查Mn 2+的内部配位范围 通过EPR和ENDOR光谱法在FosA和FosX中的硫醇选择性的稳态动力学分析; FosA和FosB，以及（iii）FosX催化的独特水合反应的机理研究。潜在 过渡态抑制剂将通过结构、光谱和动力学技术进行研究。的 底物和抑制剂与酶的相互作用的热力学将通过等温热力学来检查。 滴定量热法特别强调的是来自病原体假单胞菌的酶 铜绿假单胞菌、金黄色葡萄球菌、单核细胞增生李斯特菌和肉毒梭菌。这样做的目的是 研究的目的是建立机制和结构的基础，为设计药物，以对付这两个质粒 对磷霉素的耐药性。

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. Understanding the mechanisms of antimicrobial resistance is crucial for effective patient care in the clinic and essential for developing strategies to enhance biodefence against intentionally disseminated of pathogens. 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. 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, Listeria monocytogenes and Clostridium botulinum. Genomic and biochemical analysis from this project suggest that there are three distinct subgroups ofmetalloenzymes, 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 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 from Pseudomonas aeruginosa and its relatives FosB and FosX will be determined by X-ray crystallography. The chemical and ldnede mechanisms of catalysis will be elucidated by: (i) examination of the inner coordination sphere of Mn 2+ in FosA and FosX by EPR and ENDOR spectroscopy; (ii) a steady state kinetic analysis of the thiol selectivity of FosA and FosB, and (iii) a mechanistic study of the unique hydration reaction catalyzed by FosX. Potential transition state inhibitors will investigated by structural, spectroscopic and kinetic techniques. The thermodynamics of the interaction of substrates and inhibitors with the enzymes will be examined by isothermal titration calorimetry Particular emphasis will be placed on the enzymes from the pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum. The intent of this 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.