2,4-Dinitrotoluene (2,4-DNT) dioxygenase of Burkholderia sp. strain DNT (DDO) oxidizes 2,4-DNT to 4-methyl-5-nitrocatechol (4M5NC).  Oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2- nitrotoluene (2NT), and 4NT were enhanced here by saturation mutagenesis on codon I204 of the a subunit (DntAc) of DDO allowing the degradation of both 2,3-DNT and 2,5-DNT for the first time.  The flavoprotein 4-methyl-5-nitrocatechol monooxygenase (DntB) catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone by removing the nitro group; DntB has a very narrow substrate range. Here, error prone PCR was used to create variant DntB M22L/L380I that oxidizes two new substrates: 4-nitrophenol and 3-methyl-4-nitrophenol.  This is the first report of protein engineering for nitrite removal by a flavoprotein.  Since mixtures of pollutants are frequently encountered, a hybrid dioxygenase system was also created that degrades simultaneously 0.1 mM 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.  This is the first report to describe a dioxygenase system capable of simultaneously degrading mixtures of 24DNT and naphthalene.  In addition, we used DNA shuffling to convert the archetypal naphthalene dioxygenase of Ralstonia sp. strain U2 into a nitro-aromatic-degrading enzyme which led to the discovery of the alpha subunit catalytic residues G407 and L225 that influence the regiospecificity of Rieske non-heme-iron dioxygenases.