Mutagenesis of Klebsiella aerogenes UreG To Probe Nickel Binding and Interactions with Other Urease-Related Proteins

Abstract

UreG is a GTPase required for assembly of the nickel-containing active site of urease. Herein, a Strep-tagged Klebsiella aerogenes UreG (UreGStr) and selected site-directed variants of UreGStr 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 UreGStr 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. UreGStr binds one nickel or zinc ion per monomer (Kd = ~5 μM for each metal ion) at a binding site that includes Cys72, as shown by a 12-fold increased Kd for nickel ions using C72A UreGStr 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 UreGStr 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.

Functional and Phylogenetic Analysis of ureD in Shiga Toxin-Producing Escherichia coli

“…Consistent with previous reports, these investigators found UreD to bind both UreF and UreG in vivo. The authors demonstrate that UreD also has Ni 2ϩ -binding capability and propose, supported by their additional work on UreG (2), that there is a sequential Ni 2ϩ ion transfer from UreE to UreG to UreD to the urease apoenzyme active site. To our knowledge, it is not known which residues in UreD are involved in metal ion binding or in binding to UreF or UreG.…”

Assembly of Preactivation Complex for Urease Maturation in Helicobacter pylori

“…UreG has a highly conserved nickel-binding motif shown by mutagenesis to be essential for nickel delivery (41). In the topology of the pre-activation complex we proposed (Fig.…”

Effects of Metal on the Biochemical Properties of Helicobacter pylori HypB, a Maturation Factor of [NiFe]-Hydrogenase and Urease

“…For example, metal-dependent dimerization was observed for CooC (28,29), the ATPase involved in carbon monoxide dehydrogenase biosynthesis, but ATP hydrolysis was not dramatically affected by the addition of nickel. Furthermore, the GTPase dedicated to urease maturation, UreG, binds nickel or zinc with micromolar affinities at a site that likely includes the CxH motif found at a similar location with respect to the GTPase motifs as C106/H107 of HpHypB (5,70,71). However, the relative affinities reported for nickel versus zinc, and whether the metals affect quaternary structure, vary depending on the UreG homolog.…”