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 Shapir, N.
Right arrow Articles by Wackett, L. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shapir, N.
Right arrow Articles by Wackett, L. P.
Agricola
Right arrow Articles by Shapir, N.
Right arrow Articles by Wackett, L. P.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, May 2005, p. 2214-2220, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2214-2220.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Substrate Specificity and Colorimetric Assay for Recombinant TrzN Derived from Arthrobacter aurescens TC1

Nir Shapir,1,2,4 Charlotte Rosendahl,1,{dagger} Gilbert Johnson,1 Marco Andreina,2,3 Michael J. Sadowsky,2,3,4 and Lawrence P. Wackett1,2,3*

Department of Biochemistry, Molecular Biology and Biophysics,1 BioTechnology Institute,2 Center for Microbial and Plant Genomics,3 Department of Soil, Water & Climate, University of Minnesota, St. Paul, Minnesota 551084

Received 13 September 2004/ Accepted 16 November 2004

The TrzN protein, which is involved in s-triazine herbicide catabolism by Arthrobacter aurescens TC1, was cloned and expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified via nickel column chromatography. The purified TrzN protein was tested with 31 s-triazine and pyrimidine ring compounds; 22 of the tested compounds were substrates. TrzN showed high activity with sulfur-substituted s-triazines and the highest activity with ametryn sulfoxide. Hydrolysis of ametryn sulfoxide by TrzN, both in vitro and in vivo, yielded a product(s) that reacted with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) to generate a diagnostic blue product. Atrazine chlorohydrolase, AtzA, did not hydrolyze ametryn sulfoxide, and no color was formed by amending those enzyme incubations with NBD-Cl. TrzN and AtzA could also be distinguished by reaction with ametryn. TrzN, but not AtzA, hydrolyzed ametryn to methylmercaptan. Methylmercaptan reacted with NBD-Cl to produce a diagnostic yellow product having an absorption maximum at 420 nm. The yellow color with ametryn was shown to selectively demonstrate the presence of TrzN, but not AtzA or other enzymes, in whole microbial cells. The present study was the first to purify an active TrzN protein in recombinant form and develop a colorimetric test for determining TrzN activity, and it significantly extends the known substrate range for TrzN.

* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology and Biophysics, 140 Gortner Lab, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625-5780. E-mail: wackett{at}cbs.umn.edu.

{dagger} Present address: Department of Biology, Southern Utah University, Cedar City, UT 84720.

Applied and Environmental Microbiology, May 2005, p. 2214-2220, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2214-2220.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Seffernick, J. L., Aleem, A., Osborne, J. P., Johnson, G., Sadowsky, M. J., Wackett, L. P. (2007). Hydroxyatrazine N-Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination. J. Bacteriol. 189: 6989-6997 [Abstract] [Full Text]  
  • Shapir, N., Mongodin, E. F., Sadowsky, M. J., Daugherty, S. C., Nelson, K. E., Wackett, L. P. (2007). Evolution of Catabolic Pathways: Genomic Insights into Microbial s-Triazine Metabolism. J. Bacteriol. 189: 674-682 [Full Text]  
  • Shapir, N., Pedersen, C., Gil, O., Strong, L., Seffernick, J., Sadowsky, M. J., Wackett, L. P. (2006). TrzN from Arthrobacter aurescens TC1 Is a Zinc Amidohydrolase.. J. Bacteriol. 188: 5859-5864 [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»