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 i nclude: (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===========================================
| 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; Tronrud, Dale E; Taber, Scott R et al. (2011) Iron-containing urease in a pathogenic bacterium. Proc Natl Acad Sci U S A 108:13095-9 |
| Macomber, Lee; Hausinger, Robert P (2011) Mechanisms of nickel toxicity in microorganisms. Metallomics 3:1153-62 |
Showing the most recent 10 out of 44 publications
