Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus.

Abstract

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.

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