PROBING DRUG RESISTANCE IN B-LACTAMASE CRYSTALS BY RAMAN

提供了 本项目的主要目标是阐明导致耐药的分子因素，8- 内酰胺酶家族的酶。，8 - 1内酰胺酶由细菌产生，它们破坏青霉素型 在杀死目标细菌之前就将其转化为分子。因此，β-1内酰胺酶本身是抑制剂的靶点。 临床分离株证明，8 - 1内酰胺酶通过点突变对其药物抑制剂产生耐药性。 突变。本研究将阐明SHV-1，8-内酰胺酶与临床上常见的 重要的药物，他唑巴坦，舒巴坦和克拉维酸;这些化合物作为“自杀抑制剂”。的 反应将通过拉曼晶体学来表征-通过遵循在单晶中的反应， 使用拉曼显微镜观察酶。将药物分别注入含有晶体的母液中 在β-内酰胺酶中，抑制剂在不到一分钟的时间内完全扩散到晶体中， 可以通过拉曼差谱跟踪活性位点。使用合适形式的酶， 米氏络合物、酰基酶和最终产物的结构和群体可以在本发明的文献中定义。 晶体的拉曼数据。通过比较野生型酶的这些特性， 开发了对抑制剂的耐药性，对耐药性背后的分子机制的独特见解将 获得。8-内酰胺酶的耐药形式是M691、M69 L和M69 V，根据其临床相关性进行选择。 拉曼方法表征单晶中中间体群体的能力将用于 选择最佳的速冻时间。然后将含有捕获的反应中间体的晶体 以X射线晶体学为特征。 对于D类8-内酰胺酶OXA-10和OXA-1，最近的研究表明氨甲酰化赖氨酸起作用 在活性部位化学中的关键作用。拉曼晶体学将被用来证实这一新颖和有争议的 发现

PROVIDED. The principal goal of this project is to elucidate the molecular factors that lead to drug resistance in the ,8- lactamase family of enzymes. ,8-1actamases are produced by bacteria and they destroy penicillin-type molecules before they can kill their bacterial targets. Thus, ,8-1actamases themselves are targets for inhibitors. Clinical isolates demonstrate that ,8-1actamases develop resistance to their drug-inhibitors by undergoing point mutations. This proposal will elucidate the chemical reactions between a SHV-1 ,8-1actamase and the clinically important drugs, tazobactam, sulbactam and clavulanic acid; these compounds act as "suicide inhibitors". The reactions will be characterized by Raman crystallography - by following the reactions in single crystals of the enzyme using a Raman microscope. The drugs are injected separately into the mother liquor containing a crystal of p-lactamase, the inhibitors diffuse fully into the crystal in less than one minute and the subsequent reaction in the active site can be followed via the Raman difference spectrum. Using suitable forms of the enzyme, the structures and populations of the Michaelis complexes, acyl enzymes and final products can be defined in the crystals from the Raman data. By comparing these properties for the wild-type enzyme, and the enzyme that has developed a resistance to the inhibitors, unique insight into the molecular mechanisms underlying resistance will be gained. The resistant forms of the ,8-1actamase are M691, M69L and M69V, selected for their clinical relevance. The ability of the Raman method to characterize populations of intermediates in single crystals will be used to select optimal times for flash freezing. The crystals containing the trapped reaction intermediates will then be characterized by X-ray crystallography. For the class D ,8-1actamases OXA-10 and OXA-1 recent studies have indicated that a carbamylated lysine plays a key role in active site chemistry. Raman crystallography will be used to confirm this novel and controversial finding.