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To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone.

Results indicate that drawbacks in the degradation of dichlororinatedtoluenes is due to the unspecific attack of tetrachlorobenzene dioxygenase leading to the production of dichlorobenzaldehydes or dichlorobenzoates. The metabolic fates of the dichloromethylcatechol intermediates in the total metabolism of dichlorotoluenes are discussed.

Research has been conducted on the cis-dienelactone enzymatic transformation. The results of the (super)1 H nuclear magnetic resonance assay analysis used to study this transformation have demonstrated that that product of the cis-dienelactone hydrolase reaction is maleylacetate.

Investigates the degradation of monitoring key reactions of chloroaromatics by in situ ¹H nuclear magnetic resonance (&sup1H NMR). Use of ¹N HMR on the study of enzymatic transformation of cis-dienelactone; Maleylacetate as a product of the cis-dienelactone hydrolase reaction; Transformation of malyacetate.