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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 extract s, (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 makingmore » bio-based toluene.« less

Authors:
 [1];  [1];  [2];  [3];  [4];  [5];  [6]
+ Show Author Affiliations
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States). QB3 Inst.
  3. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biosciences
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biosciences
  5. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biosciences; Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering and of Bioengineering
  6. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth and Environmental Sciences
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1379558
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 6; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; environmental microbiology; enzymes; metabolism

Citation Formats

Zargar, K., Saville, R., Phelan, R. M., Tringe, S. G., Petzold, C. J., Keasling, J. D., and Beller, H. R. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community. United States: N. p., 2016. Web. doi:10.1038/srep31362.
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Zargar, K., Saville, R., Phelan, R. M., Tringe, S. G., Petzold, C. J., Keasling, J. D., & Beller, H. R. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community. United States. https://doi.org/10.1038/srep31362
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Zargar, K., Saville, R., Phelan, R. M., Tringe, S. G., Petzold, C. J., Keasling, J. D., and Beller, H. R. Wed . "In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community". United States. https://doi.org/10.1038/srep31362. https://www.osti.gov/servlets/purl/1379558.
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@article{osti_1379558,
title = {In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community},
author = {Zargar, K. and Saville, R. and Phelan, R. M. and Tringe, S. G. and Petzold, C. J. and Keasling, J. D. and Beller, H. R.},
abstractNote = {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 extract s, (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.},
doi = {10.1038/srep31362},
journal = {Scientific Reports},
number = 1,
volume = 6,
place = {United States},
year = {Wed Aug 10 00:00:00 EDT 2016},
month = {Wed Aug 10 00:00:00 EDT 2016}
}
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Works referenced in this record:

Catalytic Mechanism of the Glycyl Radical Enzyme 4-Hydroxyphenylacetate Decarboxylase from Continuum Electrostatic and QC/MM Calculations
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Evidence for Only Oxygenative Cleavage of Aldehydes to Alk(a/e)nes and Formate by Cyanobacterial Aldehyde Decarbonylases
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Structural Basis for a Kolbe-Type Decarboxylation Catalyzed by a Glycyl Radical Enzyme
journal, September 2011

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  • Journal of the American Chemical Society, Vol. 133, Issue 37
  • DOI: 10.1021/ja203344x

Catalytic Mechanism of the Glycyl Radical Enzyme 4-Hydroxyphenylacetate Decarboxylase from Continuum Electrostatic and QC/MM Calculations
journal, September 2013

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Anoxic hypolimnion is a significant source of biogenic toluene
journal, October 1986

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Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities
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Tolumonas auensis gen. nov., sp. nov., a Toluene-Producing Bacterium from Anoxic Sediments of a Freshwater Lake
journal, January 1996

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    Works referencing / citing this record:

    Improving methyl ketone production in Escherichia coli by heterologous expression of NADH-dependent FabG
    journal, March 2018

    • Goh, Ee-Been; Chen, Yan; Petzold, Christopher J.
    • Biotechnology and Bioengineering, Vol. 115, Issue 5
    • DOI: 10.1002/bit.26558

    Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene‐Producing Glycyl Radical Enzyme
    journal, November 2019

    • Rodrigues, Andria V.; Tantillo, Dean J.; Mukhopadhyay, Aindrila
    • ChemBioChem, Vol. 21, Issue 5
    • DOI: 10.1002/cbic.201900560

    Catabolic Pathways and Enzymes Involved in the Anaerobic Degradation of Monocyclic Aromatic Compounds
    book, January 2018

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    • Anaerobic Utilization of Hydrocarbons, Oils, and Lipids
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    Indoleacetate decarboxylase is a glycyl radical enzyme catalysing the formation of malodorant skatole
    journal, October 2018

    Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria
    journal, April 2019

    Discovery of enzymes for toluene synthesis from anoxic microbial communities
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    • DOI: 10.1038/s41589-018-0017-4

    Volatile biomarkers of symptomatic and asymptomatic malaria infection in humans
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    Volatile biomarkers of symptomatic and asymptomatic malaria infection in humans
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    Catabolic Pathways and Enzymes Involved in the Anaerobic Degradation of Monocyclic Aromatic Compounds
    book, January 2020

    • Boll, Matthias; Estelmann, Sebastian; Heider, Johann
    • Anaerobic Utilization of Hydrocarbons, Oils, and Lipids
    • DOI: 10.1007/978-3-319-50391-2_6

    Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene‐Producing Glycyl Radical Enzyme
    journal, November 2019

    • Rodrigues, Andria V.; Tantillo, Dean J.; Mukhopadhyay, Aindrila
    • ChemBioChem, Vol. 21, Issue 5
    • DOI: 10.1002/cbic.201900560

    Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria
    journal, April 2019

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