title: Identification of metal-binding residues in the Klebsiella aerogenes urease nickel metallochaperone, UreE.
abstract: The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to bind intracellular Ni(II) for transfer to urease apoprotein. While native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni, the Ni-binding sites associated with urease activation are internal to the protein as shown by studies involving truncated H144UreE [Brayman and Hausinger (1996) J. Bacteriol. 178, 5410-5416]. Nine potential Ni-binding residues (five His, two Cys, one Asp, and one Tyr) within H144UreE were independently substituted by mutagenesis to determine their roles in metal binding and urease activation. In vivo effects of these substitutions on urease activity were measured in Escherichia coli strains containing the K. aerogenes urease gene cluster with the mutated ureE genes. Several mutational changes led to reductions in specific activity, with substitution of His96 producing urease activity below the level obtained from a ureE deletion mutant. The metal-binding properties of purified variant UreE proteins were characterized by a combination of equilibrium dialysis and UV/visible, EPR, and hyperfine-shifted 1H NMR spectroscopic methods. Ni binding was unaffected for most H144UreE variants, but mutant proteins substituted at His110 or His112 exhibited greatly reduced affinity for Ni and bound one, rather than two, metal ions per dimer. Cys79 was identified as the Cu ligand responsible for the previously observed charge-transfer transition at 370 nm, and His112 also was shown to be associated with this chromophoric site. NMR spectroscopy provided clear evidence that His96 and His110 serve as ligands to Ni or Co. The results from these and other studies, in combination with prior spectroscopic findings for metal-substituted UreE [Colpas et al. (1998) J. Biol. Inorg. Chem. 3, 150-160], allow us to propose that the homodimeric protein possesses two nonidentical metal-binding sites, each symmetrically located at the dimer interface. The first equivalent of added Ni or Co binds via His96 and His112 residues from each subunit of the dimer, and two other N or O donors. Asp111 either functions as a ligand or may affect this site by secondary interactions. The second equivalent of Ni or Co binds via the symmetric pair of His110 residues as well as four other N or O donors. In contrast, the first equivalent of Cu binds via the His110 pair and two other N/O donors, while the second equivalent of Cu binds via the His112 pair and at least one Cys79 residue. UreE sequence comparisons among urease-containing microorganisms reveal that residues His96 and Asp111, associated with the first site of Ni binding, are highly conserved, while the other targeted residues are missing in many cases. Our data are most compatible with one Ni-binding site per dimer being critical for UreE's function as a metallochaperone.
authors: Department of Microbiology, Michigan State University, East Lansing 48824-1101, USA.
authors: G J
authors: GJ
authors: Colpas
authors: T G
authors: TG
authors: Brayman
authors: L J
authors: LJ
authors: Ming
authors: R P
authors: RP
authors: Hausinger
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000737:chemistry; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D014510:Urease / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D008670:Metals; D011522:Protons; D011994:Recombinant Proteins; C090404:UreE protein, Bacteria; D003035:Cobalt; D003300:Copper; D009532:Nickel; D014510:Urease