Interaction of Proteus mirabilis urease apoenzyme and accessory proteins identified with yeast two-hybrid technology

Proteus mirabilis, a gram-negative bacterium associated with complicated ur inary tract infections, produces a metalloenzyme urease which hydrolyzes ur ea to ammonia and carbon dioxide. The apourease is comprised of three struc tural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individu al UreABC heterotrimers (UreABC),, To become catalytically active, apoureas e acquires divalent nickel ions through a poorly understood process involvi ng four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains uncle ar specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed . A complex containing accessory protein UreD and structural protein UreC w as isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and U reG and structural protein UreA. In a yeast two-hybrid screen, UreD was fou nd to directly interact in vivo with coaccessory protein UreF, Unique homom ultimeric interactions of UreD and UreF were also detected in vivo. To subs tantiate the study of urease proteins with a yeast two-hybrid assay, previo usly described UreE dimers and homomultimeric UreA interactions among apour ease trimers were confirmed in vivo. Similarly, a known structural interact ion involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the ap ourease and that crucial homomultimeric associations occur among these acce ssory proteins.