Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview.Metallocenters serve essential biological functions such as transferring electrons, stabilizing biomolecules, binding substrates, and catalyzing desirable reactions. Synthesis of these sites must be tightly controlled because simple competition between metals may lead to misincorporation with loss of function and because excess cytoplasmic concentrations of free metal ions can have toxic cellular effects. In many cases, cells have evolved elaborate metallocenter assembly systems that sequester metal cofactors from the cellular milieu, thus offering protection from adventitious reactions while ensuring the fidelity of metal insertion. In addition to maintaining metal homeostasis, these assembly systems facilitate protein conformational changes and active site modifications that are required for full enzymatic activity. Several metalloproteins have been investigated as models to understand the mechanisms and dynamics of active site assembly and the complex orchestrations of their metallocenter assembly systems. In this minireview, we discuss recent findings related to maturation of the nickel-containing enzyme urease.

Protein post-translational modifications (PTMs) take many shapes, have many effects and are necessary for cellular homeostasis. One of these PTMs, Nε-lysine acetylation, was thought to occur only in the mitochondria, cytosol and nucleus, but this paradigm was challenged in the past decade with the discovery of lysine acetylation in the lumen of the endoplasmic reticulum (ER). This process is governed by the ER acetylation machinery: the cytosol:ER-lumen acetyl-CoA transporter AT-1 (also known as SLC33A1), and the ER-resident lysine acetyltransferases ATase1 and ATase2 (also known as NAT8B and NAT8, respectively). This Review summarizes the more recent biochemical, cellular and mouse model studies that underscore the importance of the ER acetylation process in maintaining protein homeostasis and autophagy within the secretory pathway, and its impact on developmental and age-associated diseases.

Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD:UreF:UreG complex which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous structurally-characterized UreH:UreF:UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 Å predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-blocking variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-down studies of cell-free extracts from tunnel-disrupting mutant cultures showed no loss of UreD interactions with urease or UreF:UreG. The nickel contents of urease samples enriched from activity-deficient cultures were decreased, while zinc and iron incorporation increased. Molecular dynamics simulations revealed size restrictions in the internal channels of the UreD variants. These findings support the role of a molecular tunnel in UreD as a direct facilitator of nickel transfer into urease, illustrating a new paradigm in active site metallocenter assembly.

The synthesis of active Klebsiella aerogenes urease via an 18-subunit enzyme apoprotein-accessory protein pre-activation complex has been well studied biochemically, but thus far this complex has remained refractory to direct structural characterization. Using ion mobility-mass spectrometry, we characterized several protein complexes between the core urease apoprotein and its accessory proteins, including the 610-kDa (UreABC)(UreDFG) complex. Using our recently developed computational modeling workflow, we generated ensembles of putative (UreABC)(UreDFG) species consistent with experimental restraints and characterized the structural ambiguity present in these models. By integrating structural information from previous studies, we increased the resolution of the ion mobility-mass spectrometry-derived models substantially, and we observe a discrete population of structures consistent with all of the available data for this complex.

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.