Mechanism of Urease Metallocenter Biosynthesis

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