Protein engineering of Rieske, non-heme dioxygenases for hydroquinone synthesis and nitrotoluene degradation

Date of Completion

January 2005


Biology, Microbiology|Chemistry, Biochemistry




Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (R34DDO) from Burkholderia cepacia R34 at position valine 350 of the α-subunit (DntAc) generated variants V350F and V350M with increased activity towards o-nitrophenol (50- and 20-fold, respectively), m-nitrophenol (30-fold), and o-methoxyphenol (170- and 150-fold, respectively) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type R34DDO has no detectable activity for these substrates). Here, the largest enhancements in V350F and V350M activity were for the synthesis of methoxyhydroquinone from o-methoxyphenol and methylhydroquinone from o-cresol. For the first time, random mutagenesis of a nitroarene dioxygenase was performed. ^ Saturation mutagenesis of the naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 at position F350 of the α-subunit (NagAc) created variant F350T that was able to degrade 2,6-dinitrotoluene (26DNT) to 3-methyl-4-nitrocatechol (3M4NC), aw well as 2-amino-4,6-dinitrotoluene (2A46DNT) to 3-amino-4-methyl-5-nitrocatechol (3A4M5NC) and 2-amino-4,6-dinitrobenzyl alcohol (2A46DNBA) (wild-type NDO has no activity on these two substrates). DNA shuffling of NDO nagAcAd generated the beneficial NagAc mutations G407S, which enhanced the reactions catalyzed by the NagAc variant F350T 3-fold, and L225R, which increased the degradation rate of 4-amino-2-nitrotoluene, generating 4-amino-2-nitrobenzyl alcohol and 4-amino-2-nitrocresol, and 23DNT, generating 2,3-dinitrobenzyl alcohol and 4-methyl-3-nitrocatechol. For the first time, random mutagenesis of NagAc discovered two new residues, G407 and L225, which influence the regiospecificity of Rieske, non-heme dioxygenases. ^ Combining two different terminal oxygenase components with single electron transfer components, we created novel hybrid dioxygenase systems for the degradation of aromatic pollutants. In the presence of naphthalene, the engineered orthric dioxygenase NDO-R34DDO (nagAa-dntAcAd-nagAbAcAd ) increased the formation rate of 4M5NC from 24DNT 2-fold relative to R34 DDO alone (NDO does not oxidize 24DNT). The construct R34DDO-NBDO ( dntAa-nbzAcAd-dntAbAcAd) generated 3A4M5NC and 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). This is the first report to describe a Rieske dioxygenase system capable of simultaneously degrading mixtures of 24DNT/naphthalene or 2A46DNT/4A26DNT, and to expand the substrate range of a Rieske dioxygenase by investigating the compatibility of the ferredoxin subunit with a dual terminal dioxygenase system. ^ Through chemical mutagenesis methods, the Burkholderia sp. strain DNT variant DNT-8 was generated with a 2-fold increase in the rate of 24DNT degradation, a 3-fold increase in the formation rate of 4M5NC, and a 2-fold increase in the nitrite release rate compared wild-type strain DNT. In addition, the variant DNT-8 was able to degrade 4M5NC 6-fold faster than wild-type DNT. This is the first report to enhance the degradation of nitroaromatic compounds through random chemical mutagenesis. ^