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Volume 149, Issue 4

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

Abstract

The tetrachlorobenzene dioxygenase (TecA) of 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 -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 of NDO and Asn of 2NTDO) were identified which control the regioselectivity of these enzymes. The corresponding amino acids at Phe and Leu 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 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.

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Keyword(s): 2NTDO, 2-nitrotoluene dioxygenase , NDO, naphthalene dioxygenase , RV, retention volume , SOE, splicing by overlap extension and TecA, tetrachlorobenzene dioxygenase
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2003-04-01
2024-04-23
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Rational engineering of the regioselectivity of TecA tetrachlorobenzene dioxygenase for the transformation of chlorinated toluenes
Microbiology 149, 903 (2003); https://doi.org/10.1099/mic.0.26054-0
/content/journal/micro/10.1099/mic.0.26054-0
/content/journal/micro/10.1099/mic.0.26054-0
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