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Applied and Environmental Microbiology, March 2006, p. 1817-1824, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1817-1824.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

Kou-San Ju and Rebecca E. Parales*

Section of Microbiology, University of California, Davis, California 95616

Received 13 September 2005/ Accepted 15 December 2005

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the {alpha} subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics.

* Corresponding author. Mailing address: Section of Microbiology, 226 Briggs Hall, 1 Shields Ave., University of California, Davis, CA 95616. Phone: (530) 754-5233. Fax: (530) 752-9014. E-mail: reparales{at}ucdavis.edu.

Applied and Environmental Microbiology, March 2006, p. 1817-1824, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1817-1824.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ferraro, D. J., Okerlund, A. L., Mowers, J. C., Ramaswamy, S. (2006). Structural basis for regioselectivity and stereoselectivity of product formation by naphthalene 1,2-dioxygenase.. J. Bacteriol. 188: 6986-6994 [Abstract] [Full Text]  


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