ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

五十年前，现代抗生素的引入开始了一场革命 在传染病的有效治疗方面。今天，这些进步在 由于越来越常见的外观，化疗处于危险之中 具有抗药性的微生物菌株。治疗的方法 越来越多的免疫缺陷个体代表着一次 必须迎接的临床挑战和选择的场所 新的、更具抗药性的菌株。目前尚不清楚肠球菌是否具有耐药性 对所有主要类别的抗生素都是一个直接的威胁 诊所。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。早些时候 在过去的十年中，观察到了质粒介导的对磷霉素的耐药性 在诊所里。其后的调查证实， 抗性质粒编码金属蛋白(FOSA或FosB)，催化 在抗生素中添加谷胱甘肽，使其失去活性。 最近，编码可能的磷霉素的染色体抗性基因 独木舟运动员已经被描述过了。这项研究项目的目标是 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药性。这些 目标包括，构建高水平的表达系统 对于蛋白质，其催化机理的阐明，以及 用X射线测定它们的三维结构 结晶学。磷霉素耐药性的调查研究 蛋白质FOSA和FOSB将包括：(1)测定动力学 预稳态动力学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；(3)测定 酶-底物相互作用在催化中的重要性；及。(Iv) 脱辅酶和脱氢酶的三维结构测定 与磷霉素结合的全酶。该项目的具体目标 关于磷霉素激酶FOSC，包括：(I)如果合成 基因和蛋白在大肠杆菌中的表达；(Ii) 酶促反应产物的结构及酶促反应 研究了该酶的动力学机制，包括 二价阳离子要求；(Iii)和对假设的检验 那个FOSC。和另一种激酶FOMA相关，以及(Iv)启动 FOSC结构的X射线结晶学研究。它是 预计这项调查将建立机制和 抗药性新药设计的结构基础 磷霉素。该项目是对计划公告的回应 PA-97-026，曲霉病、埃立克斯病与耐药性。

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.