Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease.
In vivo urease metallocenter assembly in Klebsiella aerogenes requires the presence of several accessory proteins (UreD, UreF, and UreG) and is further facilitated by UreE. In this study, UreG was isolated and shown to be a monomer with an Mr of 21,814 +/- 20 based on gel filtration chromatography and mass spectrometric results. Although it contains a P-loop motif typically found in nucleotide-binding proteins, UreG did not bind or hydrolyze ATP or GTP, and it exhibited no affinity for ATP- and GTP-linked agarose resins. Site-directed mutagenesis of ureG allowed the substitution of Ala for Lys-20 or Thr-21 in the P-loop motif and resulted in the production of inactive urease in cells grown in the presence of nickel; hence, an intact P-loop may be essential for UreG to function in vivo. These mutant cells were unable to synthesize the UreD-UreF-UreG-urease apoprotein species that are thought to be the key urease activation complexes in the cell. An insoluble protein species containing UreD, UreF, and UreG (termed the DFG complex) was detected in cells carrying deletions in ureE and the urease structural genes. The DFG complex was solubilized in 0.5% Triton X-100 detergent, shown to bind to an ATP-linked agarose resin, and found to elute from the resin in the presence of Mg-ATP. In cells containing a UreG P-loop variant, the DFG complex was formed but did not bind to the nucleotide-linked resin. These results suggest that the UreG P-loop motif may be essential for nucleotide binding by the DFG complex and support the hypothesis that nucleotide hydrolysis is required for in vivo urease metallocenter assembly.
Moncrief
M B
MB
Department of Biochemistry, Michigan State University, East Lansing 28824, USA.
Hausinger
R P
RP
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D028044:Phosphate-Binding Proteins; D011817:Rabbits; D014510:Urease / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; C086767:UreD protein, Bacteria; C101639:UreF protein, bacteria; C082270:ureG protein, Bacteria; D000255:Adenosine Triphosphate; D014510:Urease