Molecular characterization of Bacillus pasteurii UreE, a metal-binding chaperone for the assembly of the urease active site.

TitleMolecular characterization of Bacillus pasteurii UreE, a metal-binding chaperone for the assembly of the urease active site.
Publication TypeJournal Article
Year of Publication2002
AuthorsCiurli, S., N. Safarov, S. Miletti, A. Dikiy, S. K. Christensen, K. Kornetzky, D. A. Bryant, I. Vandenberghe, B. Devreese, B. Samyn, H. Remaut, and J. Van Beeumen
JournalJ Biol Inorg Chem
Date Published2002 Jun
KeywordsAmino Acid Sequence, Bacillus, Bacterial Proteins, Binding Sites, Carrier Proteins, Cloning, Molecular, Histidine, Molecular Chaperones, Molecular Sequence Data, Nickel, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Structure, Secondary, Recombinant Proteins, Sequence Homology, Amino Acid, Spectrometry, Mass, Electrospray Ionization, Urease, Zinc

The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacillus pasteurii, a novel protein putatively involved in the transport of Ni in the urease assembly process. A DNA fragment of the B. pasteurii urease operon, containing all four accessory genes (ureE, ureF, ureG, and ureD) required for the incorporation of Ni ions into the active site of urease, was cloned, sequenced, and analyzed. B. pasteurii ureE was cloned, and the UreE protein (BpUreE) was over-expressed and purified to homogeneity. The identity of the recombinant protein was determined by N- and C-terminal sequencing and by mass spectrometry. BpUreE has a chain length of 147 amino acids, and features a p I value of 4.7. As isolated, BpUreE contains one Zn(II) ion per dimer, while no Ni(II) is present, as shown by mass spectrometry and atomic absorption spectroscopy. BpUreE behaves as a dimer independently of the presence of Zn(II), as shown by gel filtration and mass spectrometry. Paramagnetic NMR spectroscopy on concentrated (2 mM) UreE solutions reveals a one Ni atom per tetramer stoichiometry, with the Ni(II) ion bound to histidines in an octahedral coordination environment. BpUreE has a high sequence similarity with UreE proteins isolated from different biological sources, while no sequence homology is observed with proteins belonging to different classes. In particular, BpUreE is most similar to UreE from Bacillus halodurans (55% identity). A multiple sequence alignment reveals the presence of four strictly conserved residues (Leu55, Gly97, Asn98, His100; BpUreE numbering), in addition to position 115, conservatively occupied by an Asp or a Glu residue. Several secondary structure elements, including a betaalphabetabetaalphabeta "ferredoxin-like" motif, are highly conserved throughout the UreE sequences.

Alternate JournalJ. Biol. Inorg. Chem.
PubMed ID12072968
Grant ListGM-31625 / GM / NIGMS NIH HHS / United States