Characterization of the Klebsiella aerogenes urease accessory protein UreD in fusion with the maltose binding protein.
Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a DeltaureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (> 670 kDa) that bound approximately 2.5 Ni2+ ions (K(d) of approximately 50 microM, where K(d) is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (approximately 4 Zn2+ ions per protomer; K(d) of 5 microM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.
Carter
Eric L
EL
Department of Microbiology and Molecular Genetics, 6193 Biomedical Physical Sciences, Michigan State University, East Lansing, MI 48824-4320, USA.
Hausinger
Robert P
RP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D004586:Electrophoresis; D021902:Enterobacter aerogenes / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D058619:Maltose-Binding Proteins; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014510:Urease / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D014510:Urease