In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.
The formation of active urease in Klebsiella aerogenes requires the presence of three structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the metallocenter in this nickel-containing enzyme. Slow and partial activation of urease apoprotein was observed after addition of nickel ion to extracts of Escherichia coli cells bearing a plasmid containing the K. aerogenes urease gene cluster or derivatives of this plasmid with deletions in ureE, ureF, or ureG. In contrast, extracts of cells containing a ureD deletion derivative failed to generate active urease, thus highlighting a key role for UreD in the metallocenter assembly process. Site-directed mutagenesis methods were used to overexpress ureD in the presence of the other urease genes, and the UreD protein was found to copurify with urease. A molecule of native urease apoprotein is capable of binding 0, 1, 2, or 3 molecules of UreD, consistent with a trimeric structure of urease catalytic units. The UreD-urease apoprotein complexes are competent for activation by nickel, with the level of activity obtained being directly related to the number of UreD molecules bound per urease molecule. Activation of the UreD-urease complexes is rapid and accompanied by UreD dissociation. We propose that UreD is a chaperone protein which stabilizes a urease apoprotein conformation that is competent for nickel incorporation.
Park
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IS
Department of Microbiology, Michigan State University, East Lansing 48824.
Carr
M B
MB
Hausinger
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RP
D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D018833:Chaperonins; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014510:Urease / Q000378:metabolism*
D001051:Apoenzymes; D001059:Apoproteins; D001426:Bacterial Proteins; D017931:DNA Primers; D008667:Metalloproteins; D011506:Proteins; C086767:UreD protein, Bacteria; D009532:Nickel; D014510:Urease; D018833:Chaperonins