Identification of Metal-Binding Residues in the Klebsiella aerogenes Urease Nickel Metallochaperon, UreE
Biochemistry 1999, 38, 4078-4088

Colpas, G. J.;1 Brayman, T. G.;1 Ming, L.-J.;2  Hausinger, R. P.*1

1Department of Microbiology and Biochemistry, Michigan State University, East Lansing, MI 48824
2Department of Chemistry and Institute for Biomolecular Science, University of South Florida, Tampa, FL 33620

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to bind intracellular Ni(II) for transfer to urease apoprotein.  Nine amino acids were chosen as potential nickel-binding residues (5 His, 2 Cys, 1 Asp, and 1 Tyr) and independently substituted by targetted mutagenesis to determine their roles in metal binding and urease activation.  In vivo effects of these substitutions on urease activity were measured in cell extracts of Escherichia coli strains containing the mutated ureE genes cloned into the K. aerogenes urease gene cluster.  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.  The results from these studies, in combination with prior spectroscopic findings for metal-substituted UreE [Colpas, G. J., Brayman, T. G., McCracken, J., Pressler, M. A., Babcock, G. T., Ming, L.-J., Colangelo, C. M., Scott, R. A., and Hausinger, R. P. (1998) J. Biol. Inorg. Chem. 3, 150-160], allow us to propose the following detailed model for the interaction of UreE with metal ions.  We suggest that the homodimeric protein possesses two nonidentical metal binding sites, each symmetrically located at the dimer interface.  Site one binds the first equivalent of Ni or Co via four histidine residues (His96 and His112 from each subunit of the dimer) and two other N or O donors (likely to include Asp111).  Site two binds the second equivalent of Ni or Co via the symmetric pair of His110 residues as well as four other N or O donors.  In contrast, the first equivalent of Cu binds to site two via the His110 pair and two other N/O donors, while the second equivalent of Cu binds at site one via the His112 pair and at least one Cys79 residue.  The latter residue is responsible for the previously observed 370 nm thiolate-to-Cu charge-transfer transition associated with this form of the protein.  UreE sequence comparisons among urease-containing microorganisms reveal that residues associated with site one Ni ligands are more highly conserved than those associated with site two ligands.  Consistent with this sequence conservation, in vivo urease activation studies indicate that only site one is critical for UreE’s function as a metallochaperone.

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