Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases

Abstract

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.

FIG. 1

Representative reduction reactions. (A) Two-electron reduction of an aromatic nitro group by a type I nitroreductase; (B) denitration of a nitroester compound by a xenobiotic reductase; (C) reduction of the α, β-unsaturated bond of 2-cyclohexen-1-one by a xenobiotic reductase.

FIG. 2

(A) Time course of the denitration of 0.9 mM NG; (B) time course of the culture density of P. putida (○), the xenA E. coli DH5α subclone (●), P. fluorescens (□), the xenB E. coli DH5α subclone (■), and E. coli DH5α harboring pUC18 without an insert (▵). P. fluorescens was incubated at 30°C; all others were incubated at 37°C.

FIG. 3

Complete nucleotide sequence and deduced amino acid sequence of the P. putida xenA gene (A) and the P. fluorescens xenB gene (B). Amino acid residues that correspond to the amino-terminal sequences determined by Edman degradation are shown in boldface type; putative −10 and −35 promoter elements and ribosome-binding sites (rbs) as well as the transcription initiation sites (+1) identified by primer extension are indicated. Inverted repeats downstream of the stop codons are denoted with arrowheads; four adenines following the xenA inverted repeat are underlined.

FIG. 4

Linear maps of putative coding regions flanking P. putida xenA (A) and P. fluorescens xenB (B). Proposed direction of transcription is shown by the arrowhead at the end of each indicated coding region. The identity of each proposed ORF is reported in Results as the top hit from a BLAST search conducted by using the deduced amino acid sequence of each indicated ORF.

FIG. 5

Denaturing gel electrophoresis analysis. (A) Comparison of 1 μg of xenobiotic reductase purified from P. putida (lane 2) to approximately 20 μg of cell extract from P. putida (lane 3), E. coli DH5α xenA subclone (lane 4), and E. coli DH5α harboring plasmid pUC18 lacking a DNA insert (lane 5); (B) comparison of 1 μg of xenobiotic reductase purified from P. fluorescens (lane 2) to approximately 20 μg of cell extract from P. fluorescens (lane 3), E. coli DH5α xenB subclone (lane 4), and E. coli DH5α harboring plasmid pUC18 lacking a DNA insert (lane 5). An arrow indicates the protein band that corresponds to the xenobiotic reductase enzyme expressed by each E. coli subclone in lanes 4. Lanes 1, molecular mass standards of 94, 67, 43, and 30 kDa.

FIG. 6

Primer extension mapping of the xenA (A) and xenB (B) transcriptional start sites. cDNA from reverse transcription reactions was loaded onto sequencing gels next to sequencing reactions (lanes GATC) conducted with the same primer. (A) Products from reactions containing 10 μg of P. putida or E. coli xenA subclone RNA are shown in lanes 1 to 4. Total RNA was isolated from P. putida (lanes 1 and 2) and from the E. coli subclone (lanes 3 and 4) after growth in LB medium (lanes 1 and 3) or LB medium supplemented with 0.9 mM NG (lanes 2 and 4). An expanded view of the nucleotide sequence surrounding the transcription initiation site (+1) is shown, and the putative −10 hexamer is highlighted with a vertical bar. (B) cDNA products from reactions containing 2 μg of E. coli xenB subclone RNA are shown in lanes 1 and 2. The nucleotide sequence surrounding the transcription initiation site (+1) is shown.

FIG. 7

Alignment of the deduced amino acid sequences of B. subtilis YqjM (Bs_YqjM), P. putida xenobiotic reductase XenA (Pp_XenA), P. fluorescens xenobiotic reductase XenB (Pf_XenB), P. syringae 2-cyclohexen-1-one reductase Ncr (Ps_Ncr), E. coli N-ethylmaleimide reductase NemA (Ec_NemA), E. cloacae PETN reductase Onr (Ecl_Onr), P. putida morphinone reductase MorB (Pp_MorB), A. radiobacter GTN reductase NerA (Ar_NerA), and S. carlsbergensis OYE isoform 1 (Sc_Oye1). Consensus of at least 50% identical amino acid residues is denoted by black boxes; conserved amino acid substitutions are highlighted with grey boxes. Amino acid residues conserved in all nine sequences are overmarked with asterisks. ▴, amino acid residues from OYE that form side chain hydrogen bonds with FMN; ▵, OYE residues that form main chain hydrogen bonds with FMN; ●, OYE active-site residue His-191.