Vitreoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo

The initial enzyme in 2,4-dinitrotoluene (2,4-DNT) catabolism from Burkholderia sp. strain DNT is DNT dioxygenase, which catalyzes oxygen addition to 2,4-DNT to form 4-methyl-5-nitrocatechol (MNC). The gene for this enzyme, dntA, was placed into Escherichia coli both in the presence and absence of the Vitreoscilla hemoglobin gene, vgb, producing strains PF6 and PFJS39, respectively. PF6 outgrew PFJS39 in LB medium and at restricted aeration in minimal medium containing 110 μM (20 ppm) 2,4-DNT. When grown in minimal medium containing 110 μM 2,4-DNT with normal aeration, the two strains converted 2,4-DNT to MNC at almost the same rate, while with restricted aeration the rate for PF6 was twice that of PFJS39. The Vmax and KM for 2,4-DNT for the conversion was determined using whole cells and whole cell lysates of both strains. For cells grown under both normal and restricted aeration in minimal/110 μM 2,4-DNT medium there was a three- to four-fold increase in Vmax for PF6 compared to PFJS39 but also about a 50% increase in the apparent KM. These results support the idea that Vitreoscilla hemoglobin can enhance bioremediation pathways of aromatic compounds that require oxygen addition at one or more steps.