Abstract
In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.
Full Text
The Full Text of this article is available as a PDF (256.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- Hartman F. C., Harpel M. R. Structure, function, regulation, and assembly of D-ribulose-1,5-bisphosphate carboxylase/oxygenase. Annu Rev Biochem. 1994;63:197–234. doi: 10.1146/annurev.bi.63.070194.001213. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- 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]
- 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]
- Maier T., Jacobi A., Sauter M., Böck A. The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein. J Bacteriol. 1993 Feb;175(3):630–635. doi: 10.1128/jb.175.3.630-635.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsudaira P. Limited N-terminal sequence analysis. Methods Enzymol. 1990;182:602–613. doi: 10.1016/0076-6879(90)82047-6. [DOI] [PubMed] [Google Scholar]
- Mobley H. L., Hausinger R. P. Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev. 1989 Mar;53(1):85–108. doi: 10.1128/mr.53.1.85-108.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Waugh R., Boxer D. H. Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity. Biochimie. 1986 Jan;68(1):157–166. doi: 10.1016/s0300-9084(86)81080-x. [DOI] [PubMed] [Google Scholar]