Mechanism of Urease Metallocenter Biosynthesis

超出所提供的空间。脲酶是一种医学上重要的酶，含有一种新的双核镍活性位点，本研究的长期目标是表征脲酶的激活机制。活性酶的细胞形成需要三种脲酶亚基（尿素、UreB和UreC）、镍离子、二氧化碳/碳酸氢盐和四种辅助蛋白（UreD、UreE、UreF和UreG）。未来的研究工作将集中在测试和完善我们提出的脲酶激活模型。我们已经证明，co2与赖氨酸侧链反应，产生氨基甲酸酯桥接两个金属离子。我们认为镍通过运输或渗透酶系统进入细胞，并通过金属伴侣蛋白UreE转运到载脂蛋白。我们认为脲酶载脂蛋白形成一个uured - uref - ureg -脲酶复合物，该复合物在gtp依赖性过程中被激活。涉及产气克雷伯菌脲酶系统的实验（研究得最好的脲酶）旨在研究我们假设的辅助蛋白的各种作用。具体目标包括：(1)进一步表征UreD-UreF-UreG-apourease复合物；(2)详细分析UreD、UreF和UreG以及这三种蛋白的复合物；(3)以及UreE的结构表征。此外，我们将(4)表征枯草芽孢杆菌（Bacillus subtilis）的脲酶激活过程，这是一种在缺乏defg同源物的情况下从脲abc簇合成脲酶的微生物。脲酶活化的研究可以作为表征金属与其他金属酶结合机制的模型系统，并将增强我们对镍这一重要微量金属离子的生物化学认识。网站性能 ======================================== 节结束 ===========================================

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===========================================