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 Keenan, B. G.
Right arrow Articles by Wood, T. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Keenan, B. G.
Right arrow Articles by Wood, T. K.

 Previous Article  |  Next Article 

Journal of Bacteriology, May 2005, p. 3302-3310, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3302-3310.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Protein Engineering of the Archetypal Nitroarene Dioxygenase of Ralstonia sp. Strain U2 for Activity on Aminonitrotoluenes and Dinitrotoluenes through Alpha-Subunit Residues Leucine 225, Phenylalanine 350, and Glycine 407

Brendan G. Keenan,1 Thammajun Leungsakul,1 Barth F. Smets,2,{dagger} Masa-aki Mori,3 David E. Henderson,4 and Thomas K. Wood1*

Departments of Chemical Engineering and Molecular and Cell Biology,1 Departments of Civil and Environmental Engineering and Molecular and Cell Biology, University of Connecticut, 191 Auditorium Road, Storrs, CT 06269-3222,2 School of Health Sciences, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka, Japan 812-8582,3 Department of Chemistry, Trinity College, 300 Summit St., Hartford, CT 061064

Received 8 December 2004/ Accepted 3 February 2005

Naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the {alpha}-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases.

* Corresponding author. Mailing address: Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, 191 Auditorium Rd., Storrs, CT 06269-3222. Phone: (860) 486-2483. Fax: (860) 486-2959. E-mail: twood{at}engr.uconn.edu.

{dagger} Present address: Environment and Resources, Technical University of Denmark, DK-2800, Lyngby, Denmark.

Journal of Bacteriology, May 2005, p. 3302-3310, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3302-3310.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Leungsakul, T., Johnson, G. R., Wood, T. K. (2006). Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from Burkholderia sp. Strain DNT for Enhanced Degradation of Nitroaromatics.. Appl. Environ. Microbiol. 72: 3933-3939 [Abstract] [Full Text]  
  • Ju, K.-S., Parales, R. E. (2006). Control of substrate specificity by active-site residues in nitrobenzene dioxygenase.. Appl. Environ. Microbiol. 72: 1817-1824 [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»