The Mechanism of Trichloroethylene Oxidation by Toluene Dioxygenase: Implications for Bioremediation
1991
Wackett, Lawrence P.
The oxidation of trichloroethylene (TCE) by toluene dioxygenase in vivo and in vitro has been investigated. In a previous study, the initial rate of TCE oxidation by Pseudomonas putida Fl declined rapidly. This was shown in vivo, in the present study, to be due to toluene dioxygenase-dependent activation of TCE to produce reactive intermediates. Carbon-14 label from metabolism of radioactive TCE was incorporated into proteins, small molecules, DNA, RNA, and lipid. Alkylation of proteins, including toluene dioxygenase, caused metabolic poisoning and the loss of TCE-degrading ability. P'. putida was able to recover from TCE-mediated cytotoxicity. Toluene dioxygenase enzyme components were purified from recombinant E. coli strains by the use of conventional chromatographic methods. The major products of TCE oxidation by purified toluene dioxygenase have been identified as formic acid and glyoxylic acid. An additional minor product was shown to result from the alkylation of reduced pyridine dinucleotide (NADPH) by a reactive TCE intermediate generated by the toluene dioxygenase catalyzed reaction. Experiments with 14C-TCE showed that enzyme inactivation was due to non-specific alkylation of the proteins by diffusible reactive intermediates. A recombinant E. coli expressing cloned toluene dioxygenase genes oxidized TCE. The rate of TCE oxidation by the E. coli strain was slower than that of P. putida Fl but the rates were sustained for a longer time. The use of recombinant strains or gratuitously induced P. putida Fl could have important implications for bioremediation. Furthermore, a greater understanding of the mechanism of toluene dioxygenase inactivation will be crucial for developing optimum systems for TCE-biotreatment.
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