title: Molecular characterization of Bacillus pasteurii UreE, a metal-binding chaperone for the assembly of the urease active site.
abstract: 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.
authors.affiliation: Department of Agro-Environmental Science and Technology, University of Bologna, Via Filippo Re 8, Italy. sciurli@agrsci.unibo.it
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
authors.forename: Niyaz
authors.initials: N
authors.lastname: Safarov
authors.forename: Silvia
authors.initials: S
authors.lastname: Miletti
authors.forename: Alexander
authors.initials: A
authors.lastname: Dikiy
authors.forename: Suzanne K
authors.initials: SK
authors.lastname: Christensen
authors.forename: Katja
authors.initials: K
authors.lastname: Kornetzky
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bryant
authors.forename: Isabel
authors.initials: I
authors.lastname: Vandenberghe
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Bart
authors.initials: B
authors.lastname: Samyn
authors.forename: Han
authors.initials: H
authors.lastname: Remaut
authors.forename: Jozef
authors.initials: J
authors.lastname: van Beeumen
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006639:Histidine / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D014510:Urease / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C090404:UreE protein, Bacteria; D006639:Histidine; D009532:Nickel; D014510:Urease; D015032:Zinc