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Journal of Bacteriology, December 2006, p. 8413-8420, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01265-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein{triangledown} ,{dagger}

Jong Kyong Kim,1 Scott B. Mulrooney,2 and Robert P. Hausinger1,2,3*

Cell and Molecular Biology Program,1 Department of Microbiology and Molecular Genetics,2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-43203

Received 10 August 2006/ Accepted 3 October 2006

Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli, a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.

* Corresponding author. Mailing address: 6193 Biomedical Physical Sciences, Michigan State University, East Lansing, MI 48824-4320. Phone: (517) 355-6463, ext. 1610. Fax: (517) 353-8957. E-mail: hausinge{at}msu.edu.

{triangledown} Published ahead of print on 13 October 2006.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, December 2006, p. 8413-8420, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01265-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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