Cloning and sequencing of the genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp. JS42

The first step in the metabolism of 2-nitrotoluene (2NT) by Pseudomonas sp. JS42 (JS42) is the addition of dioxygen to the aromatic nucleus of 2NT to form 3-methylcatechol with concomitant release of nitrite. This reaction is catalyzed by the three-component dioxygenase system 2-nitrotoluene 2,3-dioxygenase (2NTDO). We report here the cloning and nucleotide (nt) sequence of a 4912-basepair (bp) SacI DNA fragment from JS42 encoding all of the genes required for 2NTDO activity. Sequence analysis of the 4912-bp SacI DNA fragment revealed five open reading frames (ORFs). The amino acid (aa) sequences of the predicted polypeptides from these ORFs exhibit high homology to the aa sequences of polypeptides from other three-component dioxygenase systems. Based on aa sequence analyses, four of the peptides were designated Reductase2NT, Ferredoxin2NT, ISP alpha 2NT and ISP beta 2NT (ISP for iron-sulfur protein) with gene designations ntdAaAbAcAd. The predicted aa sequence from the remaining ORF (ORF2) had identity to ISP alpha subunits from other three-component dioxygenase systems but had a calculated molecular weight (M(r)) of 21,259, which is uncharacteristically small for ISP alpha subunits.