CRYSTALLOGRAPHIC STUDIES OF B-LACTAMASE

B-内酰胺酶的晶体学研究

• 批准号: 3141311
• 负责人: OSNAT HERZBERG
• 金额: $ 12.3万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3141311
• 关键词: Staphylococcus aureus; X ray crystallography; active sites; beta lactam antibiotic; beta lactamase; chemical binding; chemical kinetics; chemical stability; computer simulation; conformation; cryostat; crystallization; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; freezing; molecular dynamics; mutant; protein engineering; protein folding; protein purification; protein sequence; protein structure function; site directed mutagenesis

This application is concerned with the crystallographic aspects of a collaborative effort to study the origin of bacterial resistance to beta- lactam antibiotics related to beta-lactamase. The other parts of the collaboration include the molecular biology of beta-lactamase, in particular site directed mutagenesis, the biochemistry aspects, and the molecular modeling of the system. The study is relevant to the recovery of the effectiveness of penicillin and cephalosporin therapy, which has been rapidly diminishing due to the ability of pathogenic bacteria to produce beta-lactamases. These enzymes hydrolyze the beta-lactam ring prior to the delivery of the drug to its target - the bacterial cell wall - where the penicillin sensitive enzymes responsible for the cell wall synthesis and repair are located. The understanding of the catalytic process and the substrate specificity at the atomic level is required for the development of new effective antibiotics. X-ray crystallography provides such accurate information. The high resolution crystal structure of beta-lactamase from Staphylococcus aureus PC1 enables us to carry out binding studies to various substrates and inhibitors. Cryocrystallography, aiming at trapping reaction intermediates at liquid nitrogen temperature, will be employed, as well as conventional room temperature measurements. Site directed mutants have been designed to test the proposed catalytic mechanism, alter substrate specificity, and probe the folding properties of the enzyme. These will be prepared for biochemical and crystallographic analysis. To address the problem of the recently acquired resistance to the potent "third generation" cephalosporins, the crystal structures of the E. coli SHV1 and SHV2 beta-lactamases will be determined, the latter being responsible for the new resistance. The various aspects of the project will provide information pertaining not just to the specific clinical problem caused by beta-lactamases, but will also contribute to our fundamental understanding of protein function and stability.

该应用程序涉及晶体学方面 合作研究细菌对β-的耐药性的起源 与β-内酰胺酶相关的内酰胺抗生素。 该的其他部分 合作包括β-内酰胺酶的分子生物学， 特定位点定向诱变、生物化学方面以及 系统的分子建模。 该研究与恢复有关 青霉素和头孢菌素治疗的有效性 由于致病菌产生的能力而迅速减少 β-内酰胺酶。 这些酶在形成之前水解 β-内酰胺环。 将药物递送至其目标——细菌细胞壁——其中 负责细胞壁合成的青霉素敏感酶 维修地点。 对催化过程的理解和 开发需要原子水平的底物特异性 新的有效抗生素。 X 射线晶体学提供了如此准确的 信息。 β-内酰胺酶的高分辨率晶体结构 金黄色葡萄球菌 PC1 使我们能够进行结合研究 各种底物和抑制剂。 冷冻晶体学，旨在捕获 将使用液氮温度下的反应中间体，如 以及传统的室温测量。 定点突变体 旨在测试所提出的催化机制，改变 底物特异性，并探测酶的折叠特性。 这些将被准备用于生化和晶体学分析。 到 解决最近获得的对强效药物的抵抗力的问题 “第三代”头孢菌素，大肠杆菌的晶体结构 将测定 SHV1 和 SHV2 β-内酰胺酶，后者为 负责新的抵抗。 项目的各个方面 将提供不仅与特定临床相关的信息 β-内酰胺酶引起的问题，但也将有助于我们 对蛋白质功能和稳定性的基本了解。