Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Sep;178(18):5417–5421. doi: 10.1128/jb.178.18.5417-5421.1996

Purification and activation properties of UreD-UreF-urease apoprotein complexes.

M B Moncrief 1, R P Hausinger 1
PMCID: PMC178360  PMID: 8808930

Abstract

In vivo assembly of the Klebsiella aerogenes urease nickel metallocenter requires the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. Prior studies had shown that urease apoprotein exists in an uncomplexed form as well as in a series of UreD-urease (I.-S. Park, M.B. Carr, and R.P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S. Park and R.P. Hausinger, J. Bacteriol. 177:1947-1951, 1995) apoprotein complexes. This study demonstrates the existence of a distinct series of complexes consisting of UreD, UreF, and urease apoprotein. These novel complexes exhibited activation properties that were distinct from urease and UreD-urease apoprotein complexes. Unlike the previously described species, the UreD-UreF-urease apoprotein complexes were resistant to inactivation by NiCl2. The bicarbonate concentration dependence for UreD-UreF-urease apoenzyme activation was significantly decreased compared with that of the urease and UreD-urease apoproteins. Western blot (immunoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF. We propose that the binding of UreF modulates the UreD-urease apoprotein activation properties by excluding nickel ions from binding to the active site until after formation of the carbamylated lysine metallocenter ligand.

Full Text

The Full Text of this article is available as a PDF (405.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  2. Jabri E., Carr M. B., Hausinger R. P., Karplus P. A. The crystal structure of urease from Klebsiella aerogenes. Science. 1995 May 19;268(5213):998–1004. [PubMed] [Google Scholar]
  3. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  4. Lee M. H., Mulrooney S. B., Renner M. J., Markowicz Y., Hausinger R. P. Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J Bacteriol. 1992 Jul;174(13):4324–4330. doi: 10.1128/jb.174.13.4324-4330.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Lee M. H., Pankratz H. S., Wang S., Scott R. A., Finnegan M. G., Johnson M. K., Ippolito J. A., Christianson D. W., Hausinger R. P. Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly. Protein Sci. 1993 Jun;2(6):1042–1052. doi: 10.1002/pro.5560020617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Mobley H. L., Island M. D., Hausinger R. P. Molecular biology of microbial ureases. Microbiol Rev. 1995 Sep;59(3):451–480. doi: 10.1128/mr.59.3.451-480.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mulrooney S. B., Hausinger R. P. Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation. J Bacteriol. 1990 Oct;172(10):5837–5843. doi: 10.1128/jb.172.10.5837-5843.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mulrooney S. B., Pankratz H. S., Hausinger R. P. Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease. J Gen Microbiol. 1989 Jun;135(6):1769–1776. doi: 10.1099/00221287-135-6-1769. [DOI] [PubMed] [Google Scholar]
  9. Park I. S., Carr M. B., Hausinger R. P. In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3233–3237. doi: 10.1073/pnas.91.8.3233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Park I. S., Hausinger R. P. Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation. J Bacteriol. 1995 Apr;177(8):1947–1951. doi: 10.1128/jb.177.8.1947-1951.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Park I. S., Hausinger R. P. Metal ion interaction with urease and UreD-urease apoproteins. Biochemistry. 1996 Apr 23;35(16):5345–5352. doi: 10.1021/bi952894j. [DOI] [PubMed] [Google Scholar]
  12. Park I. S., Hausinger R. P. Requirement of carbon dioxide for in vitro assembly of the urease nickel metallocenter. Science. 1995 Feb 24;267(5201):1156–1158. doi: 10.1126/science.7855593. [DOI] [PubMed] [Google Scholar]
  13. Todd M. J., Hausinger R. P. Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes. J Biol Chem. 1987 May 5;262(13):5963–5967. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES