User:Harris.qureshi/sandbox

Overview
Urease metallocenter assembly refers to the process by which the transition metal containing active site of urease is assembled. This process includes but is not limited to the incorporation of 2 Ni2+ ions into urease, and the carbamylation of the active site lysine to render the enzyme functionally active. The pathway has been found to be GTP-dependent and facilitated via a cascade of accessory proteins which bind the apo enzyme and shuttle nickel to the resultant complex for insertion.

Unfacilitated Metallocenter Assembly
The necessity for facilitated loading of urease with Ni2+ comes from evidence that the nickel binding site is heavily buried in the protein, and not solvent exposed. Specifically, the metal cannot be removed from the enzyme using the chelating agent EDTA, unless conditions are employed to denature or unfold the protein. Moreover, urease with no nickel bound does not easily take up nickel from solution, with or without denaturing conditions. This information led to suggestions that nickel was entering urease via protein flexibility or additional factors in biological systems

Urease Accessory Proteins
In the bacteria Klebsiella aerogenes and Helicobacter pylori, urease's (UreABC apoprotein) metallocenter assembly assisted by: UreD, UreE, UreF, UreG accessory proteins. However, UreD is referred to as UreH in H. pylori. In eukaryotes that contain urease, an UreE homologue is not known. Also, despite the fact that eukaryotic urease is composed of homo-oligomers containing their own active sites and that bacterial urease is composed of multimers with 2 to 3 subunits, the metallocenter sites are the same for all ureases. These accessory protein's genes are typically downstream & upstream of urease's subunits in bacteria, with order and letter assignments changing depending on organism. The genes for UreD, UreF, and UreG homologues are not adjacent to urease's subunit genes in eukaryotes.

Metallocenter Pathway Working Models
In models of assembly for Klebsiella aerogenes, UreD, UreF, and UreG bind either to UreABC sequentially (one UreD, UreF, UreG for each of the 3 UreABC oligomers), or as the preformed heterotrimeric UreD:UreF:UreG complex. Formation of the ultimate UreABC:UreD:UreF:UreG:UreE complex is facilitated by further binding of nickel-bound UreE. The final complex's role includes GTP hydrolysis by UreG, Ni2+ insertion, and active site lysine carbamylation, with the accessory proteins dissociating from urease at the finish. Currently, the mechanism for nickel insertion and lysine carbamylation by bicarbonate is still a subject for study.

Role
It is hypothesized that UreH/D's role is to act as a scaffold that binds urease to recruit the other accessory proteins, and facilitate nickel insertion into the active site. The exact location of UreD binding to urease is not known. Modelling attempts using experimental data have shown UreD/H binding to the UreB & UreC subunits of apo urease, with one UreD/H bound to each UreABC oligomer.

Evidence to Support Role
The beta-helical fold in UreH contains 17 beta strands and 2 alpha helices. This fold resembles another scaffold complex involved in metallocenter synthesis. Additionally, a study with metal-free urease incubated with nickel and UreD led to 15% more functional urease than without UreD. Due to this information, it was hypothesized that UreH/D is involved in serving as a scaffold and or recruiting the other accessory proteins, and is involved in facilitating nickel insertion into the active site.

Role
In Klebsiella aerogenes, UreF's role is to bind UreABC near UreB to provide a binding site for UreG and to regulate UreG's ability to catalyze GTP hydrolysis. UreF exercises its regulatory role via binding interactions with the subsequently bound UreG on the opposite side of UreG's GTP site. In this way, GTP hydrolysis can be efficiently coupled to urease activation. UreD & UreF are both thought to bind close to UreB, the apoenzyme component of urease that could allow access to the forming active site via a hinge-like motion.

Evidence to Support Role
UreF inH. pylori (PDB: 3CXN) and can form alpha-helical bridging dimers that link 2 UreHs and serve as a binding domain for UreG. In this complex, a helix at the N-terminus of UreF is bound by UreH and allows for UreF's Tyr48 to become unburied. The UreG binding site is the strongly conserved UreF dimer face which contains its C-terminus and Tyr48.

Functionally, UreABC:UreD:UreF has similar urease activity to when UreF is not bound (UreABC:UreD). Since UreF was found to bind UreG on a face opposite to UreG's GTP site in a UreH:UreF:UreG complex, it is not believed to be a GTPase-activating protein because it does not permit it to enhance activity. Additionally, UreG shows enhanced GTPase activity in the complex when UreF is mutated. This information supports the idea that UreF controls GTPase activity in a way to ensure that GTP hydrolysis and metallocenter assembly are efficiently paired.

Role
The role of UreG in Klebsiella aerogenes is to bind UreABC:UreD:UreF or UreF to increase its GTPase activity such that it can hydrolyze GTP and participate in nickel insertion. UreG's Asp80 has been identified as key in this binding interaction. Note that UreG cannot bind the complex when UreB is absent, and that monomeric UreG contains a metal binding site where Zn2+ and Ni2+ have been found. Currently, a crystal structure of free monomeric UreG is not available, likely attributed to UreG being an intrinsically disordered protein.

Evidence for Role
UreG binding to apo urease (UreABC:UreD:UreF) cannot occur when it lacks UreB. Monomeric UreG also exhibits poor to no GTPase activity, as compared to when bound to the urease activation complex (UreABC:UreD:UreF:UreG). Through studying the mutagenesis of residues' effects on complex formation, Asp80 was identified as key on the UreG binding region of UreF. Additionally, when incubated with nickel and bicarbonate (for lysine carbamylation), 60% of present active sites in UreABC:UreD:UreF:UreG have nickel insertion and become active.

Monomeric UreG of ‘’K. aerogenes’’ can bind either Ni2+ or Zn2+ in a 1:1 ratio. However, the UreABC:UreD:UreF:UreG complex can form without Ni2+ in K. aerogenes, and in vitro by combining UreABC:UreD:UreF and UreG.

Role
UreE in K. aerogenes is a metallochaperone that is thought to bind cytoplasmic Ni2+ and shuttle the ions to the urease activation complex for insertion via binding interactions with UreG. The residues involved UreE-UreG interactions are not yet identified. UreE contains a C-terminal Polyhistidine-tag region that can bind 6 Ni2+ ions. Though, UreE mutants lacking this region can still contribute to urease activation. When dimerized, UreE:UreE contains an interfacial metal binding site containing His96 from each UreE, and 2 peripheral metal binding sites that included His110 & His112 from each subunit. The interfacial site appears to be the only metal binding site necessary for UreE function.

Evidence for Role
UreABC:UreD:UreF:UreG:UreE has been found to generate completely activated urease.

A UreE mutant lacking the Polyhistidine-tag (H144*UreE (PDB: 1GMW)) bound with copper, possessed 3 sites for metal binding: (1) an interfacial site containing His-96 from each UreE subunit, and (2) peripheral metal binding sites containing His110 & His112 on each UreE. Mutations in the interfacial metal binding site of UreE led to inactive UreE, as compared to mutations in other sites which did not impair function.

Mechanism for Metal Insertion
Currently, the exact mechanism of nickel insertion to urease from UreE via the activation complex UreABC:UreD:UreF:UreG:UreE is not established.