Expression, purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

•3NTDO components were expressed, purified to homogeneity and characterized.•Reconstituted 3NTDO was in vitro active and removed nitro group efficiently.•3NTDO specificity was highest for 3-NT whereas least for 2,4-DNT.•Methylcatechol formation from 3-NT reveals its degradation pathway in D. sp. DS2.•Recombinant 3NTDO also yielded chlorocatechols from chloronitro compounds. 3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.