Mechanism of Urease Metallocenter Biosynthesis

脲酶金属中心生物合成机制

• 批准号: 6888102
• 负责人: ROBERT P HAUSINGER
• 金额: $ 22.43万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6888102
• 关键词: Klebsiella; active sites; apoenzymes; bacterial proteins; carbon dioxide; enzyme biosynthesis; high performance liquid chromatography; metal metabolism; microorganism culture; microorganism metabolism; nickel; oligonucleotides; ultraviolet spectrometry; urease

EXCEED THE SPACE PROVIDED. The long-term objective of this proposal is to characterize the activation mechanism for urease, a medically important enzyme that contains a novel dinuclear nickel active site. Cellular formation of active enzyme requires three urease subunits (UreA, UreB, and UreC), nickel ions, carbon dioxide/bicarbonate, and four accessory proteins (UreD, UreE, UreF, and UreG). Future research efforts will focus on testing and refining our proposed model for urease activation. We have shown that CO2reacts with a lysine side chain and the resulting carbamate bridges the two metal ions. We propose that nickel enters the cell via a transport or permease system and is shuttled to the apoprotein by the metallochaperone UreE. We suggest that urease apoprotein forms a UreD- UreF-UreG-apourease complex that is activated in a GTP-dependent process. Experiments involving the urease system ofKlebsiella aerogenes (thebest-studied urease) are designed to investigate various roles we have hypothesized for the accessory proteins. Specific aims include: (1) further characterization of the UreD-UreF-UreG-apourease complex, (2) detailed analyses of UreD, UreF, and UreG, as well as a complex of these three proteins, (3) and structural characterization of UreE. In addition, we will (4) characterize the urease activation process in Bacillus subtilis, a microorganism that synthesizes urease from a ureABC cluster in the absence ofureDEFG homologues. This work on urease activation may serve as a model system for characterizing the mechanisms of metal incorporation into other metalloenzymes, and it will enhance our understanding of the biochemistry of nickel, an essential trace metalion. PERFORMANCE SITE ========================================Section End===========================================

超过提供的空间。该提案的长期目标是表征尿素的激活机制，尿素是一种具有新颖的二核镍活性位点的医学重要酶。活性酶的细胞形成需要三个尿素酶亚基（尿素，UREB和尿素），镍离子，二氧化碳/碳酸氢碳和四种辅助蛋白（URED，UREE，UREF和UREG）。未来的研究工作将着重于测试和完善我们提出的尿素激活模型。我们已经表明，具有赖氨酸侧链的二氧化碳和所得的氨基甲酸酯桥接两个金属离子。我们建议镍通过传输或份系统进入电池，并通过金属伴侣Uree将其穿梭至载脂蛋白。我们建议尿素酶载脂蛋白形成在依赖GTP依赖性过程中激活的Uref-ureg-poRease复合物。涉及尿布系统的实验（最佳尿素酶）旨在研究我们假设辅助蛋白的各种作用。具体目的包括：（1）进一步表征URED-uref-ureg-apourease复合物，（2）对URED，UREF和UREG的详细分析，以及这三种蛋白质的复合物，（3）和Uree的结构表征。此外，我们（4）将表征枯草芽孢杆菌中的尿素酶激活过程，枯草芽孢杆菌是一种微生物，在没有RuestEFG同源物的情况下从尿素BC簇中合成尿素。这项关于脲酶激活的工作可以用作模型系统，以表征金属掺入其他金属酶的机制，它将增强我们对镍生物化学的理解，这是必不可少的痕量金属。表演站点=============================================================================================