MECHANISM OF UREASE METALLOCENTER BIOSYNTHESIS

细菌毒力因子--活性尿素酶的合成 新的bi-Ni活性位点，需要四个辅助基因(ed，ureE，ureF， 和ureG)，以及三个编码基因(ureB和urec) 尿素酶亚基。功能合并所需的ATRID-G 镍进入酶。这个项目的长期目标是 阐明辅助蛋白在尿素酶作用机制中的作用 金属分配中心组件。这项关于尿素酶激活的工作可以作为一种 用于理解其他金属结合机理的模型系统 金属酶，包括许多医学上重要的。此外，它还将 极大地提高了我们对镍的生物化学的知识，镍是一种重要的 微量金属离子。最后，更好地理解了 这种酶中的金属分配中心组装可能与潜在的 尿路结石等尿素酶引起的临床预防方法 条件。 本项目将阐明金属分配中心组装的机制。 产气克雷伯氏菌最具特性的尿素酶。具体的 目的包括：(A)鉴定一种可能的尿素酶特异性伴侣蛋白 蛋白。将提纯Ced/尿素酶脱辅基蛋白复合体并将其 已检查属性。使用纯化的复合体，尿素酶的幅度和速率 将在存在其他辅助蛋白和 细胞因素。经纯化后的活性产物 本课程将对HERD/尿素酶脱辅基蛋白复合体进行表征。最后， 我们将阐明ATED本身的特性。(B)描述UreE，a 可能是尿素酶脱辅基蛋白的镍供体。一个真正的尿素酶缺失突变体将 以克服我们目前缺失菌株的局限性。 将使用定点突变方法来鉴定金属- 并探讨这种镍结合蛋白的作用。这个 UreE蛋白和金属分配中心的结构将进一步研究。 (C)鉴定UreG，一种可能的G蛋白。UreG在网络中的作用 在尿素酶激活过程中建立或解离各种复合体 将被检测，分离出的蛋白质将被表征和研究 定点诱变方法。特别值得关注的是 尝试在以下情况下测量UreG的GTP酶活性 适当的因素。(D)Uref的特征。UreF蛋白将 其在尿素酶金属分配中心组装中的作用将被提纯 特色化的。(E)尿素酶辅助基因缺失对 将对整个电池的镍摄取进行评估。

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.