Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases

The genes encoding flavin mononucleotide-containing oxidoreductases, design ated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroe ster bond were cloned, sequenced, and characterized. The P. putida gene, xe nA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that remo ves 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). Th e P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-depende nt flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily rea ct with 2-cyelohexen-1-one. Heterologous expression of xenA and xenB was de monstrated in Escherichia coli DH5 alpha. The transcription initiation site s of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB seq uences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryot ic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serv e a detoxification role in antioxidant defense systems.