MECHANISM OF UREASE METALLOCENTER BIOSYNTHESIS

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DESCRIPTION: The long-term objective of this proposal is to characterize the biosynthesis of urease, a medically important enzyme that contains a novel binuclear-Ni active site. Formation of active enzyme requires the presence of the three urease subunits (UreA, UreB, and UreC), four accessory gene products (UreD, UreE, UreF, and UreG), carbon dioxide, and nickel ions. The four auxiliary proteins facilitate metal ion incorporation into the apoenzyme to form functional enzyme by as yet unclear processes. The CO2 is incorporated as a carbamate of a lysine that serves as a ligand to the metal center. Dr. Hausinger will elucidate the mechanism of metallocenter assembly in the best-characterized urease, that from Klebsiella aerogenes. His specific aims include: (1) purification and characterization of a complex that is comprised of UreD, UreF, and UreG, (2) formation of large quantities of a UreD-UreF-UreG-urease apoprotein complex, (3) examination of the activation properties of the UreD-UreF-UreG-urease apoprotein, (4) characterization of the UreE metallocenter and further investigation of whether this protein functions in Ni delivery to urease apoprotein, and (5) detailed analysis of the accessory protein-free activation process for urease apoprotein. The goal is to assess whether the accessory proteins function in any of the following roles: (1) providing specificity so that only Ni is bound to the apoprotein, (b) restricting the metal binding modes so that Ni can bind only in the proper coordination geometry, (c) catalyzing expulsion of non-Ni metal ions or incorrectly bound Ni ions, (d) aiding in the generation and/or delivery of CO2, and (e) assisting in the transfer of Ni from the presumed Ni carrier, UreE, to the apoprotein. This work on urease activation may serve as a model system for characterizing the mechanisms of metal incorporation into other metalloenzymes and it will greatly enhance our understanding of the biochemistry of Ni, an essential trace metal ion.