Monitoring key reactions in degradation of chloroaromatics by in situ ¹H nuclear magnetic resonance: Solution structures of metabolites formed from cis-dienelactone

A ¹H nuclear magnetic resonance (¹H NMR) assay was used to study the enzymatic transformation of cis-dienelactone, a central intermediate in the degradation of chloroaromatics. It was shown that the product of the cis-dienelactone hydrolase reaction is maleylacetate, in which there is no evidence for the formation of 3-hydroxymuconate. Under acidic conditions, the product structure was 4-carboxymethyl-4-hydroxybut-2 -en-4-olide. Maleylacetate was transformed by maleylacetate reductase into 3-oxoadipate, a reaction competing with spontaneous decarboxylation into cis-acetylacrylate. One-dimensional ¹H NMR in ¹H₂O could thus be shown to be an excellent noninvasive tool for monitoring enzyme activities and assessing the solution structure of substrates and products.