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 i nclude: (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.
| Martin-Diaconescu, Vlad; Joseph, Crisjoe A; Boer, Jodi L et al. (2017) Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes. J Biol Inorg Chem 22:497-503 |
| Macomber, Lee; Minkara, Mona S; Hausinger, Robert P et al. (2015) Reduction of urease activity by interaction with the flap covering the active site. J Chem Inf Model 55:354-61 |
| Farrugia, Mark A; Wang, Beibei; Feig, Michael et al. (2015) Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease. Biochemistry 54:6392-401 |
| Boer, Jodi L; Mulrooney, Scott B; Hausinger, Robert P (2014) Nickel-dependent metalloenzymes. Arch Biochem Biophys 544:142-52 |
| Farrugia, Mark A; Han, Linjie; Zhong, Yueyang et al. (2013) Analysis of a soluble (UreD:UreF:UreG)2 accessory protein complex and its interactions with Klebsiella aerogenes urease by mass spectrometry. J Am Soc Mass Spectrom 24:1328-37 |
| Farrugia, Mark A; Macomber, Lee; Hausinger, Robert P (2013) Biosynthesis of the urease metallocenter. J Biol Chem 288:13178-85 |
| Boer, Jodi L; Hausinger, Robert P (2012) Klebsiella aerogenes UreF: identification of the UreG binding site and role in enhancing the fidelity of urease activation. Biochemistry 51:2298-308 |
| Carter, Eric L; Proshlyakov, Denis A; Hausinger, Robert P (2012) Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease. J Inorg Biochem 111:195-202 |
| Carter, Eric L; Boer, Jodi L; Farrugia, Mark A et al. (2011) Function of UreB in Klebsiella aerogenes urease. Biochemistry 50:9296-308 |
| Macomber, Lee; Elsey, Scott P; Hausinger, Robert P (2011) Fructose-1,6-bisphosphate aldolase (class II) is the primary site of nickel toxicity in Escherichia coli. Mol Microbiol 82:1291-300 |
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