GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins.
Syntheses of metal-containing enzymes often require the participation of accessory proteins. The roles played by many of these accessory proteins are poorly characterized. Klebsiella aerogenes urease, a nickel-containing enzyme, provides an ideal system to study metallocenter assembly. Here, we describe a method for isolating a complex containing urease apoprotein and the UreD, UreF, and UreG accessory proteins. We demonstrate that urease apoprotein in this complex is activated to near wild-type enzyme levels when incubated with nickel ions and high (approximately 100 mM) concentrations of bicarbonate. Significantly, we also observed nickel-dependent activation at physiologically relevant (approximately 100 microM) bicarbonate levels, but only in the presence of GTP. Based on studies involving a nonhydrolyzable analog of GTP, we conclude that nucleotide hydrolysis, not just binding, is required for this process. The critical nucleotide-binding site was localized to UreG on the basis of experiments using a variant complex. These studies highlight the relevance of the UreD-UreF-UreG-urease apoprotein complex to nickel metallocenter assembly and explain the previously identified in vivo energy requirement for urease activation.
Soriano
A
A
Departments of Microbiology and Biochemistry, Michigan State University, East Lansing, MI, 48824, USA.
Hausinger
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RP
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry*; D001639:Bicarbonates / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry*; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D009532:Nickel / Q000494:pharmacology; D028044:Phosphate-Binding Proteins; D014510:Urease / Q000737:chemistry* / Q000378:metabolism*
D001051:Apoenzymes; D001426:Bacterial Proteins; D001639:Bicarbonates; D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; C086767:UreD protein, Bacteria; C101639:UreF protein, bacteria; C082270:ureG protein, Bacteria; D009532:Nickel; D006160:Guanosine Triphosphate; D014510:Urease