title: Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly.
abstract: 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 beta-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 (Kd of 9.6 +/- 1.3 microM) 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.
authors.affiliation: Department of Microbiology, Michigan State University, East Lansing 48824-1101.
authors.forename: M H
authors.initials: MH
authors.lastname: Lee
authors.forename: H S
authors.initials: HS
authors.lastname: Pankratz
authors.forename: S
authors.initials: S
authors.lastname: Wang
authors.forename: R A
authors.initials: RA
authors.lastname: Scott
authors.forename: M G
authors.initials: MG
authors.lastname: Finnegan
authors.forename: M K
authors.initials: MK
authors.lastname: Johnson
authors.forename: J A
authors.initials: JA
authors.lastname: Ippolito
authors.forename: D W
authors.initials: DW
authors.lastname: Christianson
authors.forename: R P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014510:Urease / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D009532:Nickel; D014510:Urease