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J Bacteriol, March 1998, p. 1194-1199, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Enzyme Specificity of 2-Nitrotoluene 2,3-Dioxygenase from Pseudomonas sp. Strain JS42 Is Determined by the C-Terminal Region of the alpha  Subunit of the Oxygenase Component

Juanito V. Parales, Rebecca E. Parales, Sol M. Resnick,dagger and David T. Gibson*

Department of Microbiology and the Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242

Received 9 October 1997/Accepted 15 December 1997

Biotransformations with recombinant Escherichia coli expressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISPalpha ) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISPbeta ) was shown to play no role in determining the specificities of these dioxygenases.


* Corresponding author. Mailing address: Department of Microbiology and the Center for Biocatalysis and Bioprocessing, 3733 Bowen Science Building, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7980. Fax: (319) 335-9999. E-mail: david-gibson{at}uiowa.edu.

dagger Present address: Department of Microbiology, Swiss Federal Institute for Environmental Science and Technology, CH-8600. Düebendorf, Switzerland.




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