JBIC Journal of Biological Inorganic Chemistry

, Volume 7, Issue 6, pp 623–631

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

Authors

  • Stefano Ciurli
    • Department of Agro-Environmental Science and Technology, University of Bologna, Via Filippo Re 8, 40127 Bologna, Italy
  • Niyaz Safarov
    • Department of Agro-Environmental Science and Technology, University of Bologna, Via Filippo Re 8, 40127 Bologna, Italy
  • Silvia Miletti
    • Department of Agro-Environmental Science and Technology, University of Bologna, Via Filippo Re 8, 40127 Bologna, Italy
  • Alexander Dikiy
    • Department of Agro-Environmental Science and Technology, University of Bologna, Via Filippo Re 8, 40127 Bologna, Italy
  • Suzanne K. Christensen
    • Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
  • Katja Kornetzky
    • Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
  • Donald A. Bryant
    • Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
  • Isabel Vandenberghe
    • Laboratory for Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, University of Gent, Gent, Belgium
  • Bart Devreese
    • Laboratory for Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, University of Gent, Gent, Belgium
  • Bart Samyn
    • Laboratory for Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, University of Gent, Gent, Belgium
  • Han Remaut
    • Laboratory for Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, University of Gent, Gent, Belgium
  • Jozef Van Beeumen
    • Laboratory for Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, University of Gent, Gent, Belgium
Original Article

DOI: 10.1007/s00775-002-0341-7

Cite this article as:
Ciurli, S., Safarov, N., Miletti, S. et al. J Biol Inorg Chem (2002) 7: 623. doi:10.1007/s00775-002-0341-7

Abstract.

The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacilluspasteurii, 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. pasteuriiureE 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 pI 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 Bacillushalodurans (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 βαββαβ "ferredoxin-like" motif, are highly conserved throughout the UreE sequences. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0341-7.

Metal-binding chaperone Urease Nickel Bacillus pasteurii

Copyright information

© SBIC 2002