Cloning and expression of ntnD, encoding a novel NAD(P)(+)-independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp strain TW3

Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrob enzoate and toluene to benzoate aerobically via a route analogous to the up per pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenza ldehyde and benzaldehyde, respectively. The gene is located downstream of t he previously reported ntn gene cluster. NtnD bears no similarity to the an alogous TOL plasmid XyIB (benzyl alcohol dehydrogenase) protein either in i ts biochemistry, being NAD(P)(+) independent and requiring assay via dye-li nked electron transfer, or in its deduced amino acid sequence. It does, how ever, have significant similarity in its amino acid sequence to other NAD(P )(+)-independent alcohol dehydrogenases and contains signature patterns cha racteristic of type III flavin adenine dinucleotide-dependent alcohol oxida ses. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same tran script as the other ntn genes. The substrate specificity of the enzyme expr essed from the cloned and overexpressed gene was similar to the activity ex pressed from strain TW3 grown on 4-nitrotoluene, providing evidence that nt nD is the previously unidentified gene in the pathway of 4-nitrotoluene cat abolism. Examination of the 14.8-kb region around the ntn genes suggests th at one or more recombination events have been involved in the formation of their current organization.