Rational engineering of the regioselectivity of TecA tetrachlorobenzene dioxygenase for the transformation of chlorinated toluenes.

The tetrachlorobenzene dioxygenase (TecA) of Ralstonia sp. PS12 carries out the first step in the aerobic biodegradation of chlorinated toluenes. Besides dioxygenation of the aromatic ring of 4-chloro-, 2,4-, 2,5- and 3,4-dichlorotoluene as the main reaction, it also catalyses mono-oxygenation of the methyl groups of 2,3-, 2,6-, 3,5-di- and 2,4,5-trichlorotoluene as the main reactions, channelling these compounds into dead-end pathways. Based on the crystal structure of the homologous naphthalene dioxygenase (NDO) and alignment of the alpha-subunits of NDO and TecA, the substrate pocket of TecA was modelled. Recently, for NDO and the homologous 2-nitrotoluene dioxygenase (2NTDO), two amino acids (Phe(352) of NDO and Asn(258) of 2NTDO) were identified which control the regioselectivity of these enzymes. The corresponding amino acids at Phe(366) and Leu(272) of TecA were substituted to change the regioselectivity and to expand the product spectrum. Position 366 was shown to control regioselectivity of the enzyme, although mutations resulted in decreased or lost activity. Amino acid substitutions at Leu(272) had little or no effect on the regioselectivity of TecA, but had significant effects on the product formation rate. Substitutions at both positions changed the site of oxidation of 2,4,5-trichlorotoluene slightly. As new products, 3,4,6-trichloro-1-methyl-1,2-dihydroxy-1,2-dihydrocyclohexan-3,5-diene, 4,6-dichloro-3-methylcatechol, 3,6-dichloro-4-methylcatechol and 3,4-dichloro-6-methylcatechol were identified.