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 Ekberg, M.
Right arrow Articles by Sjöberg, B.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ekberg, M.
Right arrow Articles by Sjöberg, B.-M.

 Previous Article  |  Next Article 

Journal of Bacteriology, February 2003, p. 1167-1173, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1167-1173.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Vivo Assay for Low-Activity Mutant Forms of Escherichia coli Ribonucleotide Reductase

Monica Ekberg,{dagger} Pernilla Birgander, and Britt-Marie Sjöberg*

Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden

Received 16 July 2002/ Accepted 18 November 2002

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.

* Corresponding author. Mailing address: Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden. Phone: 46-8-164150. Fax: 46-8-166488. E-mail: Britt-Marie.Sjoberg{at}molbio.su.se.

{dagger} Present address: Accuro Immunology AB, SE-171 77 Stockholm, Sweden.

Journal of Bacteriology, February 2003, p. 1167-1173, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1167-1173.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Fujimitsu, K., Su'etsugu, M., Yamaguchi, Y., Mazda, K., Fu, N., Kawakami, H., Katayama, T. (2008). Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli. J. Bacteriol. 190: 5368-5381 [Abstract] [Full Text]  
  • Chang, M. C. Y., Yee, C. S., Stubbe, J., Nocera, D. G. (2004). Turning on ribonucleotide reductase by light-initiated amino acid radical generation. Proc. Natl. Acad. Sci. USA 101: 6882-6887 [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»