PROBING DRUG RESISTANCE IN B-LACTAMASE CRYSTALS BY RAMAN

项目描述（由申请人提供）：本项目的主要目标是阐明导致b -内酰胺酶家族耐药的分子因素。(-内酰胺酶是由细菌产生的，它们在杀死目标细菌之前破坏青霉素类分子。因此，b -内酰胺酶本身是抑制剂的靶标。临床分离表明，b -内酰胺酶通过发生点突变对其药物抑制剂产生耐药性。阐明SHV-1 b -内酰胺酶与临床重要药物他唑巴坦、舒巴坦、克拉维酸之间的化学反应；这些化合物起到了“自杀抑制剂”的作用。这些反应将用拉曼晶体学来表征——用拉曼显微镜跟踪酶的单晶反应。将药物单独注射到含有b -内酰胺酶晶体的母液中，抑制剂在不到1分钟的时间内完全扩散到晶体中，通过拉曼差谱可以跟踪活性部位的后续反应。利用适当形式的酶，可以根据拉曼数据在晶体中定义米切里斯配合物、酰基酶和最终产物的结构和居群。通过比较野生型酶和对抑制剂产生抗性的酶的这些特性，将获得对抗性分子机制的独特见解。b -内酰胺酶的耐药形式是M691、M69L和M69V，选择它们是因为它们的临床相关性。拉曼方法表征单晶中间体居群的能力将用于选择闪冷冻的最佳时间。然后用x射线晶体学对含有捕获反应中间体的晶体进行表征。对于D类b -内酰胺酶OXA-10和OXA-1，最近的研究表明氨甲酰赖氨酸在活性位点化学中起关键作用。拉曼晶体学将用于证实这一新颖而有争议的发现。

DESCRIPTION (provided by applicant): The principal goal of this project is to elucidate the molecular factors that lead to drug resistance in the B-lactamase family of enzymes. (-lactamases are produced by bacteria and they destroy penicillin-type molecules before they can kill their bacterial targets. Thus, B-lactamases themselves are targets for inhibitors. Clinical isolates demonstrate that, B-lactamases develop resistance to their drug-inhibitors by undergoing point mutations. This proposal will elucidate the chemical reactions between a SHV-1 B-lactamase 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 B-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 B-lactamase 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 B-lactamases 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.