(442g) Orthric Rieske Dioxygenases for Degrading Aromatic Pollutants

Since mixtures of pollutants are frequently encountered, a hybrid dioxygenase system was created here that degrades simultaneously 0.1 mM 2,4-dinitrotoluene (24DNT) and 0.1 mM naphthalene by combining the terminal oxygenase genes (dntAcAd) of the 2,4-dinitrotoluene dioxygenase (R34DDO) from Burkholderia cepacia R34 with the electron transport and terminal oxygenase genes (nagAaAbAcAd) of the naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 (NDO-R34DDO). Neither NDO nor R34DDO alone had significant activity on 24DNT or naphthalene, respectively. NDO-R34DDO also degraded 0.1 mM 4-nitrotoluene 5-fold faster than R34DDO alone and 1.5-fold faster than NDO alone, indicating that the novel NDO-R34DDO dioxygenase could also oxidize additional substrates with high proficiency. The terminal oxygenase genes (nbzAcAd) of the nitrobenzene dioxygenase (NBDO) from Comamonas sp. strain JS765 were also combined with the dntAaAbAcAd genes of R34DDO, generating R34DDO-NBDO, and the dntAcAd genes of DNTDDO from Burkholderia sp. strain DNT were combined with the nagAaAb genes of NDO and nbzAcAd genes of NBDO, generating NDO-NBDO-DNTDDO, to create hybrid dioxygenases with the ability to simultaneously degrade the 2,4,6-trinitrotoluene (TNT) reduction products 2-amino-4,6-dinitrotoluene (2A46DNT) and 4A26DNT. During simultaneous incubation with 0.25 mM 2A46DNT and 0.25 mM 4A26DNT, R34DDO-NBDO generated 3-amino-4-methyl-5-nitrocatechol (3A4M5NC) and 2-amino-4,6-dinitrobenzyl alcohol (2A46DNBA) from 2A46DNT 4-fold and 3-fold faster, respectively, than NBDO alone and formed 3A6M5NC from 4A26DNT (R34DDO alone has no activity on 4A26DNT). Therefore, the addition of NBDO nbzAcAd to R34DDO allows for a more balanced degradation of the TNT reduction products. The novel construct NDO-NBDO-DNTDDO generated 3A6M5NC from 4A26DNT 3-fold faster than NBDO alone. This is the first report to describe a dioxygenase system capable of simultaneously degrading mixtures of 24DNT and naphthalene and mixtures of 2A46DNT and 4A26DNT and to investigate the compatibility of the ferredoxin subunit from a Rieske non-heme iron dioxygenase with a dual terminal dioxygenase system.