Controlling regiospecific oxidation of aromatics and the degradation of chlorinated aliphatics via active site engineering of toluene monooxygenases

Toluene monooxygenases from pseudomonads are powerful enzymes whose activities have not been fully appreciated. Recently the literature was corrected to show there was no true, predominantly meta-hydroxylating enzyme, and it was discovered that these enzymes perform two and three successive hydroxylations of benzene. The physiological relevance of these successive hydroxylations by toluene p-monooxygenase (TpMO) was discerned for the toluene degradation pathway of Ralstonia pickettii PKO1. More importantly, these successive hydroxylations create the possibility of producing substituted aromatics for industrially important syntheses. To take advantage of this potential and to probe the hydroxylation reaction itself, DNA shuffling has been used to determine the residues that control the regiospecific catalytic activity. This regiospecific activity was fine tuned using saturation mutagenesis to create monooxygenases that, along with the wild-type enzymes, are able to form various doubly hydroxylated, substituted aromatics including 3-nitrocatechol, 4-nitrocatechol, nitrohydroquinone, methylhydroquinone, 4-methylresorcinol, methoxyresorcinol, 3-methoxycatechol, 2-naphthol, and 1-hydroxyfluorene. The regiospecific control of hydroxylation has reached the point where toluene may be hydroxylated at the ortho-, meta-, and para-positions by altering just two amino acids near the diiron active site, and these residues may be altered to create the first predominantly meta-hydroxylating enzyme. Also, regiospecific oxidation of naphthalene may be controlled to form both 1- and 2-naphthol (first report of microbial formation of this compound), and the regiospecific oxidation of indole may be controlled so that a single enzyme [toluene o-monooxygenase (TOM) of Burkholderia cepacia G4] may be used to form a variety of indigoids including predominantly indigo, isoindigo, indirubin, and isatin. To control the successive hydroxylations, two-phase reactors have also been designed to produce phenol from benzene and 2-naphthol from naphthalene using toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1. To show the utility of these enzymes for pathway engineering, they have been combined with both glutathione S-transferases as well as epoxide hydrolases to create engineered bacteria that have accelerated degradation rates for chlorinated ethenes as well as to take advantage of the fact that only one oxygenase has been shown to oxidize tetrachloroethylene (toluene o-xylene monooxygenase of Pseudomonas stutzeri OX1). Similar monooxygenation reactions for substituted aromatics have been discovered using dinitrotoluene dioxygenases.