| Abstract
| - The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to bindintracellular Ni(II) for transfer to urease apoprotein. While native UreE possesses a histidine-rich regionat its carboxyl terminus that binds several equivalents of Ni, the Ni-binding sites associated with ureaseactivation are internal to the protein as shown by studies involving truncated H144*UreE [Brayman andHausinger (1996) J. Bacteriol. 178, 5410−5416]. Nine potential Ni-binding residues (five His, two Cys,one Asp, and one Tyr) within H144*UreE were independently substituted by mutagenesis to determinetheir roles in metal binding and urease activation. In vivo effects of these substitutions on urease activitywere measured in Escherichia coli strains containing the K. aerogenes urease gene cluster with the mutatedureE genes. Several mutational changes led to reductions in specific activity, with substitution of His96producing urease activity below the level obtained from a ureE deletion mutant. The metal-bindingproperties of purified variant UreE proteins were characterized by a combination of equilibrium dialysisand UV/visible, EPR, and hyperfine-shifted 1H NMR spectroscopic methods. Ni binding was unaffectedfor most H144*UreE variants, but mutant proteins substituted at His110 or His112 exhibited greatly reducedaffinity for Ni and bound one, rather than two, metal ions per dimer. Cys79 was identified as the Culigand responsible for the previously observed charge-transfer transition at 370 nm, and His112 also wasshown to be associated with this chromophoric site. NMR spectroscopy provided clear evidence thatHis96 and His110 serve as ligands to Ni or Co. The results from these and other studies, in combinationwith 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-bindingsites, each symmetrically located at the dimer interface. The first equivalent of added Ni or Co binds viaHis96 and His112 residues from each subunit of the dimer, and two other N or O donors. Asp111 eitherfunctions as a ligand or may affect this site by secondary interactions. The second equivalent of Ni or Cobinds via the symmetric pair of His110 residues as well as four other N or O donors. In contrast, the firstequivalent of Cu binds via the His110 pair and two other N/O donors, while the second equivalent of Cubinds 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 Nibinding, are highly conserved, while the other targeted residues are missing in many cases. Our data aremost compatible with one Ni-binding site per dimer being critical for UreE's function as a metallochaperone.
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