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Purification and characterization of Klebsiella aerogenes UreE protein: A nickel‐binding protein that functions in urease metallocenter assembly
Author(s) -
Lee Mann Hyung,
Pankratz H. Stuart,
Wang Shengke,
Scott Robert A.,
Finnegan Michael G.,
Johnson Michael K.,
Ippolito Joseph A.,
Christianson David W.,
Hausinger Robert P.
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560020617
Subject(s) - nickel , chemistry , crystallography , urease , octahedron , biochemistry , enzyme , crystal structure , organic chemistry
The Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174 , 4324‐4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic β ‐strand and exhibits unusually tight binding to phenyl‐Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule ( M r = 35, 000) binds 6.05 + 0.25 nickel ions ( K d of 9.6 + 1.3 μ M) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X‐ray absorption and variable‐temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo‐octahedral geometry with 3‐5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X‐ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.