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Applied and Environmental Microbiology, November 2000, p. 4742-4750, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from Pseudomonas fluorescens I-C

Jeong W. Pak,1 Kyle L. Knoke,1,dagger Daniel R. Noguera,2,* Brian G. Fox,3 and Glenn H. Chambliss1

Department of Bacteriology,1 Department of Civil and Environmental Engineering,2 and Department of Biochemistry,3 University of Wisconsin, Madison, Wisconsin 53706

Received 8 May 2000/Accepted 11 August 2000

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-14C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2,6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic m/z of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of ~0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O2, but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP+. Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The m/z 376 dimers also reacted with either dansyl chloride or N-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the m/z 376 species to various isomers of amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.

* Corresponding author. Mailing address: Department of Civil and Environmental Engineering, 1415 Engineering Drive, Madison, WI 53706. Phone: (608) 263-7783. Fax: (608) 262-5199. E-mail: noguera{at}engr.wisc.edu.

dagger Present address: Dupont Experimental Station, Wilmington, DE 19880-0328.

Applied and Environmental Microbiology, November 2000, p. 4742-4750, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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