title: Klebsiella aerogenes UreF: identification of the UreG binding site and role in enhancing the fidelity of urease activation.
abstract: The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a UreD-UreF-UreG complex bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. This study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein-protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in copurification of UreD and urease apoprotein, whereas UreG bound to only a subset of the species. Notably, weakened interaction with UreG correlated with the low-activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD-UreF-UreG-urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the noncognate metal Zn.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States.
authors.forename: Jodi L
authors.initials: JL
authors.lastname: Boer
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D021902:Enterobacter aerogenes / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D014510:Urease / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014510:Urease