Mutagenesis of Klebsiella aerogenes UreG to probe nickel binding and interactions with other urease-related proteins.
UreG is a GTPase required for assembly of the nickel-containing active site of urease. Herein, a Strep-tagged Klebsiella aerogenes UreG (UreG(Str)) and selected site-directed variants of UreG(Str) were constructed for studying the in vivo effects on urease activation in recombinant Escherichia coli cells, characterizing properties of the purified proteins, and analysis of in vivo and in vitro protein-protein interactions. Whereas the Strep tag had no effect on UreG's ability to activate urease, enzyme activity was essentially abolished in the K20A, D49A, C72A, H74A, D80A, and S111A UreG(Str) variants, with diminished activity also noted with E25A, C28A, and S115A proteins. Lys20 and Asp49 are likely to function in binding/hydrolysis of GTP and binding of Mg, respectively. UreG(Str) binds one nickel or zinc ion per monomer (K(d) approximately 5 microM for each metal ion) at a binding site that includes Cys72, as shown by a 12-fold increased K(d) for nickel ions using C72A UreG(Str) and by a thiolate-to-nickel charge-transfer band that is absent in the mutant protein. Based on UreG homology to HypB, a GTPase needed for hydrogenase assembly, along with the mutation results, His74 is likely to be an additional metal ligand. In vivo pull-down assays revealed Asp80 as critical for stabilizing UreG(Str) interaction with the UreABC-UreDF complex. In vitro pull-down assays demonstrated UreG binding to UreE, with the interaction enhanced by nickel or zinc ions. The metallochaperone UreE is suggested to transfer its bound nickel to UreG in the UreABC-UreDFG complex, with the metal ion subsequently transferring to UreD and then into the nascent active site of urease in a GTP-dependent process.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Jodi L
JL
Boer
Soledad
S
Quiroz-Valenzuela
Kimberly L
KL
Anderson
Robert P
RP
Hausinger
D001665:Binding Sites / Q000235:genetics; D004754:Enterobacter / Q000235:genetics / Q000378:metabolism; D021902:Enterobacter aerogenes / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism; D007709:Klebsiella; D056511:Metallochaperones; D008670:Metals; D016296:Mutagenesis; D009532:Nickel / Q000378:metabolism*; D011506:Proteins / Q000235:genetics; D014510:Urease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc
D056511:Metallochaperones; D008670:Metals; D011506:Proteins; D009532:Nickel; D006160:Guanosine Triphosphate; D006864:Hydrogenase; D014510:Urease; D015032:Zinc