Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus.
Klebsiella aerogenes UreE, one of four accessory proteins involved in urease metallocenter assembly, contains a histidine-rich C terminus (10 of the last 15 residues) that is likely to participate in metal ion coordination by this nickel-binding protein. To study the function of the histidine-rich region in urease activation, ureE in the urease gene cluster was mutated to result in synthesis of a truncated peptide, H144* UreE, lacking the final 15 residues. Urease activity in cells containing H144* UreE approached the activities for cells possessing the wild-type protein at nickel ion concentrations ranging from 0 to 1 mM in both nutrient-rich and minimal media. In contrast, clear reductions in urease activities were observed when two ureE deletion mutant strains were examined, especially at lower nickel ion concentrations. Surprisingly, the H144* UreE, like the wild-type protein, was readily purified with a nickel-nitrilotriacetic acid resin. Denaturing polyacrylamide gel electrophoretic analysis and N-terminal sequencing confirmed that the protein was a truncated UreE. Size exclusion chromatography indicated that the H144* UreE peptide associated into a homodimer, as known for the wild-type protein. The truncated protein was shown to cooperatively bind 1.9 +/- 0.2 Ni(II) ions as assessed by equilibrium dialysis measurements, compared with the 6.05 +/- 0.25 Ni ions per dimer reported previously for the native protein. These results demonstrate that the histidine-rich motif is not essential to UreE function and is not solely responsible for UreE nickel-binding ability. Rather, we propose that internal nickel binding sites of UreE participate in urease metallocenter assembly.
Brayman
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TG
Department of Biochemistry, Michigan State University, East Lansing 48824-1101, USA.
Hausinger
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RP
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009532:Nickel / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017384:Sequence Deletion; D013053:Spectrophotometry; D014510:Urease / Q000187:drug effects / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D010446:Peptide Fragments; C090404:UreE protein, Bacteria; D006639:Histidine; D003300:Copper; D009532:Nickel; D014510:Urease