Purification and Properties of the Klebsiella aerogenes UreE Metal-Binding Domain, a Functional Metallochaperone of Urease

Abstract

Klebsiella aerogenes UreE, a metallochaperone that delivers nickel ions during urease activation, consists of distinct "peptide-binding" and "metal-binding" domains and a His-rich C terminus. Deletion analyses revealed that the metal-binding domain alone is sufficient to facilitate urease activation. This domain was purified and shown to exhibit metal-binding properties similar to those of UreE lacking only the His-rich tail.

The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein

“…The metal binding properties of the UreEF fusion protein were further characterized by UV-visible spectroscopy and compared to the spectra of metal-bound wild-type UreE. Many studies have focused on spectroscopic metal binding analysis of H144* UreE that lacks the His-rich carboxyl terminus (3,6,8,25), but no UV-visible studies have been reported on wild-type UreE until this analysis. As illustrated by Fig.…”

“…The resulting plasmid (pTBEF-GCins), containing a translational fusion of the ureE and ureF genes, was digested with BstXI and AatII and was cloned into similarly digested pKK17 (7) to yield pKK-EF (containing the entire K. aerogenes urease operon, but with ureE and ureF fused). pKK-⌬E and pKK-⌬F (containing the complete urease operon with ureE or ureF deleted) were generated by the subcloning procedures described in Table 1, using pACT-KK⌬2-136f (25) and pKAU17⌬ureF L2 (16), respectively. pET-EF was constructed by cloning the BamHI-AatII fragment of pTBEFGCins into similarly digested pETH144*⌬G, resulting in the insertion of the ureEF fusion gene into pET21 for purification of the fusion protein.…”

“…This multistep process is guided by the action of several accessory proteins that, in bacteria, are typically encoded by genes located in the same cluster as the structural genes. Klebsiella aerogenes possesses the best-studied urease activation system involving the ureDABCEFG urease gene cluster, in which UreD, UreF, and UreG form a GTPdependent molecular chaperone (37) and UreE functions as a metallochaperone by delivering nickel to the urease apoprotein (UreABC) (8,25,36). Among these accessory proteins, only UreE and UreG are highly soluble, resulting in the publication of many studies to characterize these proteins (primarily using the recombinant K. aerogenes proteins produced in Escherichia coli or the native proteins from Bacillus pasteurii) at a biochemical and structural level (7,8,21,26,32,35,39).…”

Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins

“…Synthesis of active urease requires the action of several accessory proteins (23), with the best-studied system found in Klebsiella aerogenes, in which the structural genes are found in a gene cluster containing four accessory genes (ureDABCEFG). By use of this system, UreD-UreF-UreG was identified as a GTPdependent molecular chaperone that binds urease apoprotein (8, 32), while UreE was shown to function as a metallochaperone that delivers Ni 2ϩ (11,25,31). Genome sequence analysis has revealed that, in contrast to other ureolytic microorganisms, Bacillus subtilis contains only urease structural genes (ureABC) and lacks homologues to any accessory genes (18).…”

Chaperones of Nickel Metabolism

“…The N-terminal domains have structural similarities to a domain of the yeast Hsp40 molecular chaperone Sis1 [81], suggesting that this domain may be involved in molecular recognition and binding to other urease accessory proteins and/or urease apoprotein. Arguing against this conclusion are results from studies involving a construct that produced only the metal-binding domain of K. aerogenes UreE (residues 70-143); the single domain of UreE is capable of delivering Ni to the urease apoprotein and the N-terminal domain is not required [82].…”