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-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper 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-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)(+) independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)(+)-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization.
James
K D
KD
School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, Wales, United Kingdom.
Hughes
M A
MA
Williams
P A
PA
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D019905:Benzyl Alcohol / Q000378:metabolism; D001592:Benzyl Alcohols / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014050:Toluene / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D001592:Benzyl Alcohols; D011994:Recombinant Proteins; D014050:Toluene; D009249:NADP; C046885:4-nitrobenzyl alcohol; C017572:4-nitrotoluene; D000429:Alcohol Oxidoreductases; C084444:NtnD protein, Pseudomonas sp. TW3; D019905:Benzyl Alcohol