The UreEF fusion protein provides a soluble and functional form of the UreF urease accessory protein.
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.
Kim
Jong Kyong
JK
Cell and Molecular Biology Program, Michigan State University, East Lansing, MI 48824-4320, USA.
Mulrooney
Scott B
SB
Hausinger
Robert P
RP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D021902:Enterobacter aerogenes / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017353:Gene Deletion; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014510:Urease / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C090404:UreE protein, Bacteria; C101639:UreF protein, bacteria; D014510:Urease