title: In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community.
abstract: 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 O2, 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.
authors: Joint BioEnergy Institute (JBEI), 5885 Hollis Avenue, Emeryville, CA, USA.
authors: K
authors: K
authors: Zargar
authors: Joint BioEnergy Institute (JBEI), 5885 Hollis Avenue, Emeryville, CA, USA.
authors: R
authors: R
authors: Saville
authors: Joint BioEnergy Institute (JBEI), 5885 Hollis Avenue, Emeryville, CA, USA.|QB3 Institute, University of California, Berkeley, California 94270, United States.
authors: R M
authors: RM
authors: Phelan
authors: Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, USA.|Biosciences, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, USA.
authors: S G
authors: SG
authors: Tringe
authors: Joint BioEnergy Institute (JBEI), 5885 Hollis Avenue, Emeryville, CA, USA.|Biosciences, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, USA.
authors: C J
authors: CJ
authors: Petzold
authors: Joint BioEnergy Institute (JBEI), 5885 Hollis Avenue, Emeryville, CA, USA.|Biosciences, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, USA.|Departments of Chemical &Biomolecular Engineering and of Bioengineering, University of California, Berkeley, CA, USA.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé, DK2970-Hørsholm, Denmark.
authors: J D
authors: JD
authors: Keasling
authors: Joint BioEnergy Institute (JBEI), 5885 Hollis Avenue, Emeryville, CA, USA.|Earth and Environmental Sciences, LBNL, Berkeley, CA, USA.
authors: H R
authors: HR
authors: Beller
mesh_terms: D000577:Amides / Q000737:chemistry; D000693:Anaerobiosis; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000378:metabolism*; D002384:Catalysis; D002474:Cell-Free System; D003013:Clostridium; D007218:Industrial Microbiology; D010100:Oxygen / Q000737:chemistry; D016133:Polymerase Chain Reaction; D040901:Proteomics; D012336:RNA, Ribosomal, 16S; D012722:Sewage; D014050:Toluene / Q000737:chemistry*
chemical_list: D000577:Amides; D012336:RNA, Ribosomal, 16S; D012722:Sewage; D014050:Toluene; C425067:4-hydroxyphenylacetate decarboxylase; D002262:Carboxy-Lyases; D010100:Oxygen