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Journal of Bacteriology, October 1999, p. 6254-6263, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cloning and Sequence Analysis of Two Pseudomonas Flavoprotein Xenobiotic Reductases

David S. Blehert,1 Brian G. Fox,2,* and Glenn H. Chambliss1,*

Department of Bacteriology, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706,1 and Institute for Enzyme Research and Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 537052

Received 21 May 1999/Accepted 30 July 1999

The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P. putida gene, xenA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of xenA and xenB was demonstrated in Escherichia coli DH5alpha . The transcription initiation sites of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems.

* Corresponding author. Mailing address for Brian G. Fox: Institute for Enzyme Research, 1710 University Ave., University of Wisconsin, Madison, WI 53705. Phone: (608) 262-9708. Fax: (608) 265-2904. E-mail: fox{at}enzyme.wisc.edu. Mailing address for Glenn H. Chambliss: Department of Bacteriology, Rm. 225 E. B. Fred Hall, University of Wisconsin, Madison, WI 53706. Phone: (608) 262-1161. Fax: (608) 262-9865. E-mail: ghchambl{at}facstaff.wisc.edu.

Journal of Bacteriology, October 1999, p. 6254-6263, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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