In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE

Abstract

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni2+; however, a truncated form of this protein (H144*UreE) binds only 2 Ni2+ per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni2+ to spectinomycin-treated Escherichia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni2+ per dimer with elevated K(d) values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni2+ per dimer with unperturbed K(d) values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999) Biochemistry 38, 4078-4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni2+ with k1 ~18 and k2 ~100 M-1 s-1, and with dissociation rates k-1 ~3 x 10-3 and k-2 ~10-4 s-1. Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni2+ do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation.