ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

五十年前，现代抗生素的引入引发了一场革命 有效治疗传染病。 今天这些进步 由于越来越常见的出现，化疗正处于危险之中 微生物的耐药菌株。 治疗 越来越多的免疫功能低下的个体立即代表 必须应对的临床挑战以及选择的场所 新的、更具抵抗力的菌株。 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构成直接威胁 诊所。 磷霉素是一种强效、广谱抗生素，有效 针对革兰氏阳性和革兰氏阴性微生物。 早在 过去十年观察到质粒介导的磷霉素耐药性 在诊所里。 随后的调查证实， 抗性质粒编码催化金属蛋白（FosA 或 FosB） 在抗生素中添加谷胱甘肽，使其失去活性。 最近，编码假定磷霉素的染色体抗性基因 已经描述了独木舟者。 本研究项目的目标 阐明酶的催化机制和结构 参与微生物对磷霉素的耐药性。 这些 目标包括，构建高级表达系统 对于蛋白质，阐明其催化机制，以及 通过 X 射线测定其三维结构 晶体学。 磷霉素耐药性的研究 蛋白质 FosA 和 FosB 将包括： (i) 动力学测定 预稳态和稳态动力学催化机理 技术； (ii) 阐明催化的化学机制 包括金属离子在反应中的作用 磁共振（EPR 和 ENDOR）技术； (三) 决定 催化中重要的酶-底物相互作用； (四) 脱辅酶三维结构的测定和 与磷霉素结合的全酶。 项目的具体目标 关于磷霉素激酶 FosC，包括： (i) 合成，如果 大肠杆菌中该蛋白的基因和表达； (ii) 确定 酶促反应产物的结构 研究酶的动力学机制，包括 二价阳离子需求； (iii) 和假设检验 那个 FosC。和另一种激酶 FomA 相关，并且 (iv) 启动 FosC 结构的 X 射线晶体学研究。 它是 预计这项调查将确定机制和 设计抗耐药性新药的结构基础 磷霉素。 该项目是对计划公告的回应 PA-97-026，曲霉病、埃利希体病和耐药性。

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.