MECHANISM OF UREASE METALLOCENTER BIOSYNTHESIS

脲酶金属中心生物合成机制

• 批准号: 2144889
• 负责人: ROBERT P HAUSINGER
• 金额: $ 11.71万
• 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 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144889
• 关键词: G protein; Klebsiella; RNA binding protein; X ray crystallography; X ray spectrometry; active sites; apoenzymes; bacterial genetics; bacterial proteins; biological transport; enzyme activity; enzyme biosynthesis; enzyme complex; gene deletion mutation; guanosinetriphosphatases; metal metabolism; metalloproteins; molecular chaperones; nickel; nuclear magnetic resonance spectroscopy; nucleic acid sequence; protein sequence; protein structure function; site directed mutagenesis; urease

Synthesis of active urease, a bacterial virulence factor containing a novel bi-Ni active site, requires four accessory genes (ureD, ureE, ureF, and ureG) in addition to the three genes (ureA, ureB, and ureC) encoding the urease subunits. UreD-G are required for functional incorporation of Ni into the enzyme. The long-term objective of this project is to elucidate the role of the accessory proteins in the mechanism of urease metallocenter assembly. This work on urease activation may serve as a model system for understanding the metal incorporation mechanisms of other metalloenzymes, including many of medical importance. Moreover, it will greatly enhance our knowledge of the biochemistry of Ni, an essential trace metal ion. Finally, an improved understanding of the mechanism for metallocenter assembly in this enzyme may have relevance to potential methods for prevention of urinary stones and other urease-induced clinical conditions. This project will elucidate the mechanism of metallocenter assembly in the best-characterized urease, that from Klebsiella aerogenes. The specific aims include: (a) Characterize UreD, a possible urease-specific chaperonin protein. The UreD/urease apoprotein complex will be purified and its properties examined. Using purified complex, the extent and rate of urease activation will be studied in the presence of other accessory proteins and cellular factors. Products resulting from activation of the purified UreD/urease apoprotein complex will be characterized. Finally, the properties of UreD itself will be elucidated. (b) Characterize UreE, a possible Ni donor to urease apoprotein. A true ureE deletion mutant will be generated to overcome the limitations of our current deletion strains. Site-directed mutagenesis methods will be used to identify the metal- binding ligands and probe the role of this Ni-binding protein. The structure of the UreE protein and metallocenter will be further examined. (c) Characterize UreG, a possible G protein. The function of UreG in establishing or dissociating various complexes during urease activation will be examined and isolated protein will be characterized and studied by site-directed mutagenesis methods. Of particular interest will be attempts to measure GTPase activity for UreG in the presence of appropriate factors. (d) Characterization of UreF. The UreF protein will be purified and its function in urease metallocenter assembly will be characterized. (e) The effects of urease accessory gene deletions on nickel uptake by whole cells will be assessed.

活性尿素酶的合成，一种含有A的细菌毒力因子 新型BI-NI活性位点需要四个附属基因（URE，UREE，UREF， 除了编码三个基因（尿素，UREB和尿素）外 尿素亚基。 URED-G是功能合并所必需的 ni进入酶。该项目的长期目标是 阐明辅助蛋白在脲酶机理中的作用 金属中心组件。这项有关尿素激活的工作可能是 了解其他金属掺入机制的模型系统 金属酶，包括许多医学重要性。 而且，它将 大大增强了我们对NI生物化学的了解，这是必不可少的 痕量金属离子。最后，改善了对机制的理解 在该酶中的金属中心装配可能与潜力有关 预防尿石和其他尿素酶诱导的临床的方法 状况。 该项目将阐明金属中心装配的机制 最佳特征尿素酶，来自克雷伯氏菌的尿素。具体 目的包括：（a）特征是，可能的尿素特异性伴侣蛋白 蛋白质。 URED/URESE酶载蛋白复合蛋白复合物将被纯化并 检查的属性。使用纯化的复合物，尿素的程度和速率 将在存在其他附件蛋白的存在下研究激活 细胞因子。纯化的激活产生的产品 将表征URED/脲酶载脂蛋白复合物。最后， 您的特性本身将被阐明。 （b）特征uree，a 可能的NI供体尿酶载脂蛋白。真正的Uree删除突变体将 生成以克服当前缺失菌株的局限性。 位置定向的诱变方法将用于识别金属 结合配体并探测该Ni结合蛋白的作用。 这 Uree蛋白质和金属中心的结构将进一步研究。 （c）表征Ureg，可能的G蛋白。 Ureg在 在激活过程中建立或解离各种复合物 将检查并通过 定向诱变方法。 特别有趣的是 尝试在存在的情况下测量UREG的GTPase活性 适当的因素。 （d）UREF的表征。 UREF蛋白将 纯化，其在尿素金属中心组件中的功能将是 特征。 （e）尿素辅助基因缺失对 将评估全细胞的镍吸收。