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

Abstract

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 monooxygenation 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 a-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.

Chloromethylmuconolactones as Critical Metabolites in the Degradation of Chloromethylcatechols: Recalcitrance of 2-Chlorotoluene

“…To elucidate whether chloromethylcatechols can be transformed by chlorocatechol pathway enzymes and, specifically, whether intermediary substituted muconates can be subjected to dehalogenation, a mixture of 3-chloro-4-methylcatechol (8%), 4-chloro-3-methylcatechol (29%), and 2-chlorobenzylalcohol (63%) was prepared from 2-chlorotoluene and a mixture of 3-chloro-5-methylcatechol (2%), 5-chloro-3-methylcatechol (18%), and 3-chlorobenzylalcohol (80%) was prepared from 3-chlorotoluene by the use of E. coli DH5␣ (pSTE44) containing the tetrachlorobenzene dioxygenase tecA1A2A3A4 and chlorobenzene dihydrodiol dehydrogenase tecB open reading frames (33) originating from Ralstonia sp. strain PS12 as previously described (35) (Fig. 1).…”

“…strain PS12 [34], were used). The relative amounts of 3-chlorobenzylalcohol and chloromethylcatechols formed by E. coli DH5␣ (pSTE44) (35) are given in parentheses. The relative amounts of lactones formed were calculated on the basis of their abundance after transformation by chlorocatechol dioxygenase and chloromuconate cycloisomerase, taking into account 3-chlorobenzylalcohol formation.…”

“…Product formation was analyzed with a Shimadzu HPLC system (LC-10AD liquid chromatograph, DGU-3A degasser, SPD-M10A diode array detector, and FCV-10AL solvent mixer) equipped with a SC125/Lichrospher column (Bischoff, Leonberg, Germany) (5 m). For analyzing substrate transformation, the aqueous solvent system (flow rate, 1 ml/min) contained 0.01% (vol/vol) H 3 PO 4 (87%) and 50% (vol/vol) methanol as previously described (35). For analysis of metabolites, the methanol concentration was reduced to 25%.…”

“…Mixtures of 3-chloro-4-methyl-and 4-chloro-3-methylcatechol and of 3-chloro-5-methyl-and 5-chloro-3-methylcatechol were prepared from 2-chlorotoluene and 3-chlorotoluene, respectively, by the use of E. coli (pSTE44) as previously described (35).…”

“…Similarly, toluene dioxygenase of P. putida F1 was observed to catalyze predominantly monooxygenation of the side chain of 2-and 3-chlorotoluene (22). The optimization of chlorotoluene degradation by rational engineering of the regioselectivity of toluene dioxygenases to avoid monooxygenation was therefore suggested (35).…”