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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 DH5
. 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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