In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p -hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p -hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p -hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O 2 , and (iv) both activities were similarly inhibited by an amide analog of p -hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.