title: Function of UreB in Klebsiella aerogenes urease.
abstract: Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreBΔ1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreBΔ1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, United States.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Carter
authors.forename: Jodi L
authors.initials: JL
authors.lastname: Boer
authors.forename: Mark A
authors.initials: MA
authors.lastname: Farrugia
authors.forename: Nicholas
authors.initials: N
authors.lastname: Flugga
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Towns
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D021902:Enterobacter aerogenes / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D058619:Maltose-Binding Proteins / Q000378:metabolism; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D015202:Protein Engineering; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014510:Urease / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D058619:Maltose-Binding Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D009532:Nickel; D010447:Peptide Hydrolases; D014510:Urease