Summary Urease, the first enzyme to be crystallized, contains a dinuclear nickel metallocenter that catalyzes the decomposition of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel versus other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel metallochaperones and GTP-dependent molecular chaperones or play other roles in the maturation process. In addition, some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.

This review describes the functions, structures, and mechanisms of nine nickel-containing enzymes: glyoxalase I, acireductone dioxygenase, urease, superoxide dismutase, [NiFe]-hydrogenase, carbon monoxide dehydrogenase, acetyl-coenzyme A synthase/decarbonylase, methyl-coenzyme M reductase, and lactate racemase. These enzymes catalyze their various chemistries by using metallocenters of diverse structures, including mononuclear nickel, dinuclear nickel, nickel-iron heterodinuclear sites, more complex nickel-containing clusters, and nickel-tetrapyrroles. Selected other enzymes are active with nickel, but the physiological relevance of this metal specificity is unclear. Additional nickel-containing proteins of undefined function have been identified.

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.

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.

Urease from Klebsiella aerogenes is composed of three subunits (UreA, UreB, and UreC) which 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 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 plus UreC to form (UreABC*)3 apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to authentic urease apoprotein, active enzyme is produced by incubation of (UreABC*)3 with Ni2+ 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 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 Ni2+ 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.