ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

50年前，现代抗生素的引入引发了一场革命。 传染病的有效治疗。 今天，这些进步 化疗是在危险中，由于越来越常见的外观 耐药微生物菌株。 治疗 越来越多的免疫功能低下的个体同时代表着 一个必须面对的临床挑战和一个选择 新的更耐药的菌株 目前尚不清楚肠球菌耐药 对所有主要抗生素类别都是直接威胁， 诊所 磷霉素是一种有效的广谱抗生素， 抗革兰氏阳性和革兰氏阴性微生物。 年初 近10年来，质粒介导的磷霉素耐药 在诊所里 随后的调查确定， 抗性质粒编码金属蛋白（FosA或FosB）， 在抗生素中加入谷胱甘肽使其失去活性。 最近，编码假定磷霉素的染色体抗性基因 独木舟运动员被描述。 本研究项目的目标 是为了阐明酶的催化机制和结构 参与微生物对磷霉素的抗性。 这些 目标包括，高水平表达系统的构建 对于蛋白质，阐明其催化机制， 用X射线测定它们的三维结构 结晶学 磷霉素耐药性调查 蛋白质FosA和FosB将包括：（i）动力学的测定， 准稳态和稳态动力学催化机理 技术;（ii）阐明催化的化学机理 包括金属离子在反应中的作用， 磁共振（EPR和ENDOR）技术;（iii）确定 在催化作用中重要的酶-底物相互作用;及（iv） 测定脱辅基酶的三维结构， 结合磷霉素的全酶。 项目的具体目标 关于磷霉素激酶FosC的合成包括：（i）如果 基因和蛋白质在大肠杆菌中的表达;（ii）确定 酶促反应产物的结构和 研究了酶的动力学机制，包括 二价阳离子要求;（iii）和假设的检验 这个Fosc。和另一种激酶FomA相关，以及（iv）启动 FosC结构的X射线晶体学研究。 是 预计这项研究将建立机械和 抗耐药性新药设计的结构基础 磷霉素。 该项目是对计划宣布的回应 PA-97-026，大肠杆菌病、埃里希体病和耐药性。

Five decades ago the introduction modern antibiotics began a revolution in effective therapy for infectious diseases. Today those advances in chemotherapy are in jeopardy due to the increasingly common appearance of drug resistant strains of microorganisms. The treatment of increasing numbers of immunocompromised individuals represents at once a clinical challenge that must be met and a venue for the selection of new, more resistant strains. It is not clear that enterococci resistant to all major classes of antibiotics are an immediate threat in the clinic. Fosfomycin is a potent, broad-spectrum antibiotic effective against both Gram-positive and Gram-negative microorganisms. Early in the last decade plasmid-mediated resistance to fosfomycin was observed in the clinic. Subsequent investigations established that the resistance plasmids encode metalloproteins (FosA or FosB) that catalyze the addition of glutathione to the antibiotic, rendering it inactive. More recently, chromosomal resistance genes encoding putative fosfomycin canoeists have been described. The objectives of this research project are to elucidate the catalytic mechanisms, and structures of enzymes involved in the resistance of microorganisms to fosfomycin. These objectives include, the construction of high-level expression systems for the proteins, the elucidation of their catalytic mechanisms, and the determination of their three-dimensional structures by X-ray crystallography. The investigations of the fosfomycin resistance proteins FosA and FosB will include: (i) a determination of the kinetic mechanism of catalysis by pre-steady state and steady state kinetic techniques; (ii) elucidation of chemical mechanism of catalysis including the role of the role of the metal ion in the reaction by magnetic resonance (EPR and ENDOR) techniques; (iii) the determination of the enzyme-substrate interactions important in catalysis; and (iv) determination of the three-dimensional structures of the apoenzyme and the holoenzyme with bound Fosfomycin. The specific aims of the project with respect to the fosfomycin kinase FosC include: (i) synthesis if the gene and expression of the protein in E.coli; (ii) a determination of the structure of the product of the enzymatic reaction and an investigation of the kinetic mechanism of the enzyme including the divalent cation requirement; (iii) and examination of the hypothesis that FosC. and another kinase FomA are related and (iv) initiation of X-ray crystallographic investigations of the structure of FosC. It is anticipated that this investigation will establish the mechanistic and structural foundation for design of new drugs to counter resistance to fosfomycin. The project is a response to program announcement PA-97-026, Aspergillosis, Ehrlichioses and Drug Resistance.