This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bessette, P. H.
Right arrow Articles by Georgiou, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bessette, P. H.
Right arrow Articles by Georgiou, G.

 Previous Article  |  Next Article 

Journal of Bacteriology, February 2001, p. 980-988, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.980-988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Effect of Sequences of the Active-Site Dipeptides of DsbA and DsbC on In Vivo Folding of Multidisulfide Proteins in Escherichia coli

Paul H. Bessette,1 Ji Qiu,2 James C. A. Bardwell,3 James R. Swartz,4 and George Georgiou1,2,*

Department of Chemical Engineering,1 and Institute for Cell and Molecular Biology,2 University of Texas, Austin, Texas 78712; Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-10483; and Department of Chemical Engineering, Stanford University, Stanford, California 94305-50254

Received 15 September 2000/Accepted 20 November 2000

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

* Corresponding author. Mailing address: Department of Chemical Engineering, University of Texas, Austin, TX 78712-1062. Phone: (512) 471-6975. Fax: (512) 471-7963. E-mail: gg{at}mail.che.utexas.edu.

Journal of Bacteriology, February 2001, p. 980-988, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.980-988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Masip, L., Klein-Marcuschamer, D., Quan, S., Bardwell, J. C. A., Georgiou, G. (2008). Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant. J. Biol. Chem. 283: 840-848 [Abstract] [Full Text]  
  • Quan, S., Schneider, I., Pan, J., Von Hacht, A., Bardwell, J. C. A. (2007). The CXXC Motif Is More than a Redox Rheostat. J. Biol. Chem. 282: 28823-28833 [Abstract] [Full Text]  
  • Segatori, L., Murphy, L., Arredondo, S., Kadokura, H., Gilbert, H., Beckwith, J., Georgiou, G. (2006). Conserved Role of the Linker {alpha}-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB. J. Biol. Chem. 281: 4911-4919 [Abstract] [Full Text]  
  • Elton, T. C., Holland, S. J., Frost, L. S., Hazes, B. (2005). F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues. J. Bacteriol. 187: 8267-8277 [Abstract] [Full Text]  
  • Kim, J.-Y., Fogarty, E. A., Lu, F. J., Zhu, H., Wheelock, G. D., Henderson, L. A., DeLisa, M. P. (2005). Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli. Appl. Environ. Microbiol. 71: 8451-8459 [Abstract] [Full Text]  
  • Tan, J., Lu, Y., Bardwell, J. C. A. (2005). Mutational Analysis of the Disulfide Catalysts DsbA and DsbB. J. Bacteriol. 187: 1504-1510 [Abstract] [Full Text]  
  • Segatori, L., Paukstelis, P. J., Gilbert, H. F., Georgiou, G. (2004). Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways. Proc. Natl. Acad. Sci. USA 101: 10018-10023 [Abstract] [Full Text]  
  • Horibe, T., Gomi, M., Iguchi, D., Ito, H., Kitamura, Y., Masuoka, T., Tsujimoto, I., Kimura, T., Kikuchi, M. (2004). Different Contributions of the Three CXXC Motifs of Human Protein-disulfide Isomerase-related Protein to Isomerase Activity and Oxidative Refolding. J. Biol. Chem. 279: 4604-4611 [Abstract] [Full Text]  
  • Zhan, X., Gao, J., Jain, C., Cieslewicz, M. J., Swartz, J. R., Georgiou, G. (2004). Genetic Analysis of Disulfide Isomerization in Escherichia coli: Expression of DsbC Is Modulated by RNase E-Dependent mRNA Processing. J. Bacteriol. 186: 654-660 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»