Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly.
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.
Lee
M H
MH
Department of Microbiology, Michigan State University, East Lansing 48824-1101.
Pankratz
H S
HS
Wang
S
S
Scott
R A
RA
Finnegan
M G
MG
Johnson
M K
MK
Ippolito
J A
JA
Christianson
D W
DW
Hausinger
R P
RP
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
D001426:Bacterial Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D009532:Nickel; D014510:Urease